U.S. patent number 8,143,008 [Application Number 12/771,324] was granted by the patent office on 2012-03-27 for method of nucleic acid amplification.
This patent grant is currently assigned to Illumina, Inc.. Invention is credited to Laurent Farinelli, Eric H. Kawashima, Pascal Mayer.
United States Patent |
8,143,008 |
Kawashima , et al. |
March 27, 2012 |
Method of nucleic acid amplification
Abstract
A nucleic acid molecule can be annealed to an appropriate
immobilized primer. The primer can then be extended and the
molecule and the primer can be separated from one another. The
extended primer can then be annealed to another immobilized primer
and the other primer can be extended. Both extended primers can
then be separated from one another and can be used to provide
further extended primers. The process can be repeated to provide
amplified, immobilized nucleic acid molecules. These can be used
for many different purposes, including sequencing, screening,
diagnosis, in situ nucleic acid synthesis, monitoring gene
expression, nucleic acid fingerprinting, etc.
Inventors: |
Kawashima; Eric H. (Geneva,
CH), Farinelli; Laurent (Vevey, CH), Mayer;
Pascal (Geneva, CH) |
Assignee: |
Illumina, Inc. (San Diego,
CA)
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Family
ID: |
27451621 |
Appl.
No.: |
12/771,324 |
Filed: |
April 30, 2010 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20110045541 A1 |
Feb 24, 2011 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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12148133 |
Apr 16, 2008 |
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10449010 |
Jun 2, 2003 |
7985565 |
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09402277 |
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PCT/GB98/00961 |
Apr 1, 1998 |
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Foreign Application Priority Data
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Apr 1, 1997 [GB] |
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9706528.8 |
Apr 1, 1997 [GB] |
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9706529.6 |
Jun 23, 1997 [GB] |
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9713236.9 |
Jun 23, 1997 [GB] |
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9713238.5 |
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Current U.S.
Class: |
435/6.12;
536/24.3; 536/23.1; 435/91.2; 435/287.2; 435/6.1; 536/24.33;
435/6.11; 435/91.1 |
Current CPC
Class: |
C12Q
1/6834 (20130101); C12Q 1/6869 (20130101); C12Q
1/6874 (20130101); C12N 15/1065 (20130101); C12Q
1/6837 (20130101); C12Q 1/6853 (20130101); C12Q
1/686 (20130101); C12Q 1/6837 (20130101); C12Q
2565/543 (20130101); C12Q 1/6869 (20130101); C12Q
2565/518 (20130101); C12Q 2565/537 (20130101); C12Q
2535/101 (20130101); C12Q 1/6869 (20130101); C12Q
2565/518 (20130101); C12Q 2565/537 (20130101); C12Q
2535/101 (20130101); C12Q 1/6834 (20130101); C12Q
2565/543 (20130101) |
Current International
Class: |
C12Q
1/68 (20060101); C07H 21/04 (20060101); C12P
19/34 (20060101); C12M 1/34 (20060101); C07H
21/02 (20060101) |
Field of
Search: |
;435/6,91.1,91.2,91.51,183,287.1,287.2 ;436/94,501
;536/23.1,24.3,24.33,25.3,25.32 |
References Cited
[Referenced By]
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WO 00/75374 |
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Primary Examiner: Lu; Frank W
Attorney, Agent or Firm: Thomas; Tiffany B.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of co-pending U.S. Ser. No.
12/148,133, filed on Apr. 16, 2008, which is a divisional of U.S.
Ser. No. 10/449,010, filed on Jun. 2, 2003, now U.S. Pat. No.
7,985,565 B2, which is a continuation of U.S. Ser. No. 09/402,277,
filed on Sep. 30, 1999, now abandoned, which is a .sctn.371 of
PCT/GB98/00961, filed on Apr. 1, 1998, expired. These applications
are incorporated by reference herein in their entireties
Claims
What is claimed is:
1. A method for amplifying nucleic acid molecules comprising: a)
providing a plurality of immobilized primers and immobilized first
single stranded nucleic acid molecules; b) allowing the immobilized
first single stranded nucleic acid molecules to anneal to primers
in the plurality of immobilized primers; c) extending the primers
that are annealed to the immobilized first single stranded nucleic
acid molecules using the immobilized first single stranded nucleic
acid molecules as template to provide second immobilized nucleic
acid molecules; d) separating the template and second immobilized
nucleic acid molecules using a chemical denaturant; e) allowing the
template and second immobilized nucleic acid molecules to anneal to
primers in the plurality of immobilized primers; f) extending the
primers that are annealed to the template and second immobilized
nucleic acid molecules in suitable conditions; and g) repeating
steps d)-f) to generate multiple copies of the nucleic acid
molecules.
2. The method of claim 1, wherein the first single stranded nucleic
acid molecules and primers are immobilized on a solid surface.
3. The method of claim 2, wherein the primers are substantially
homogenously dispersed over the solid surface.
4. The method of claim 2, wherein the method produces a plurality
of colonies on the solid support, wherein each colony represents a
plurality of nucleic acid molecules comprising the same
sequence.
5. The method of claim 1, wherein, prior to step a), (i)
non-immobilized single stranded nucleic acid molecules are annealed
to primers in the plurality of immobilized primers; (ii) the
annealed primers are extended using the annealed single stranded
nucleic acid molecules as template to produce immobilized nucleic
acid molecules; and (iii) the annealed single stranded nucleic acid
molecules are separated from the immobilized nucleic acid molecules
to produce the immobilized first single stranded nucleic acid
molecules of step (a).
6. The method of claim 5, wherein the non-immobilized single
stranded nucleic acid molecules have different sequences.
7. The method of claim 1, wherein the plurality of immobilized
primers comprise at least two different sequences.
8. The method of claim 7, wherein the immobilized single stranded
nucleic acid molecules comprise a first, second and third part and
wherein the first and second parts are located at the 5' and 3'
ends of the immobilized single stranded nucleic acid molecules.
9. The method of claim 8, wherein the third part is located between
the first and second parts.
10. The method of claim 9, wherein the third part is an unknown
nucleic acid sequence.
11. The method of claim 10, wherein the first part hybridizes to a
primer of one sequence in the plurality of immobilized primers and
the second part hybridizes to a primer of a different sequence in
the plurality of immobilized primers.
12. The method of claim 7, wherein in the plurality of immobilized
primers the two different sequences are present in substantially
the same concentrations.
13. The method of claim 1, further comprising releasing one or more
of the template or second immobilized nucleic acid molecules.
14. The method of claim 1, further comprising determining the
sequence of one or more of the nucleic acid molecules of step
(g).
15. The method of claim 14, wherein the second immobilized nucleic
acid molecules are released and the sequence of the template is
determined.
16. The method of claim 15, wherein the sequencing is performed by
extending primers hybridized to the template.
17. The method of claim 16, wherein the sequencing is performed
using labeled nucleotides.
18. The method of claim 17, wherein the label is a fluorescent
label.
19. The method of claim 1, wherein the immobilized single stranded
nucleic acid molecules of step (a) are different from one
another.
20. The method of claim 19, wherein the immobilized primers
comprise the same sequence.
21. The method of claim 20, wherein the method produces a plurality
of different nucleic acid molecules.
22. The method of claim 21, wherein the sequence of the plurality
of different nucleic acid molecules is determined in parallel.
23. The method of claim 1, wherein the chemical denaturant is an
organic solvent.
24. The method of claim 23, wherein the organic solvent is
formamide.
Description
This invention relates, inter alia, to the amplification of nucleic
acids.
Molecular biology and pharmaceutical drug development now make
intensive use of nucleic acid analysis (Friedrich, G. A. Moving
beyond the genome projects, Nature Biotechnology 14, 1234 (1996)).
The most challenging areas are whole genome sequencing, single
nucleotide polymorphism detection, screening and gene expression
monitoring. Currently, up to hundreds of thousands of samples are
handled in single DNA sequencing projects (Venter, J. C., H. O
Smith, L. Hood, A new strategy for genome sequencing, Nature 381,
364 (1996)). This capacity is limited by the available technology.
Projects like the "human genome project" (gene mapping and DNA
sequencing) and identifying all polymorphisms in expressed genes
involved in common diseases imply the sequencing of millions of DNA
samples.
With most of the current DNA sequencing technologies, it is simply
not possible to decrease indefinitely the time required to process
a single sample. A way of increasing throughput is to perform many
processes in parallel. The introduction of robotic sample
preparation and delivery, 96 and 384 well plates, high density
gridding machines (Maier, E., S. Meierewer, A. R. Ahmadi, J.
Curtis, H. Lehrach, Application of robotic technology to automated
sequence fingerprint analysis by oligonucleotide hybridization,
Journal Of Biotechnology 35, 191 (1994)) and recently the
development of high density oligonucleotide arrays (Chee, M., R.
Yang, E. Hubbell, A. Berno, X. C. Huang, D. Stern, J. Winkler, D.
J. Lockhart, M. S. Morris, and S. P. A. Fodor, Accessing genetic
information with high-density DNA arrays, Science
274(5287):610-614, (1996)) are starting to bring answers to the
demand in ever higher throughput. Such technologies allow up to
50,000.about.100,000 samples at a time to be processed within days
and even hours (Maier, E., Robotic technology in library screening,
Laboratory Robotics and Automation 7, 123 (1995)).
In most known methods for performing nucleic acid analysis, it is
necessary to first extract the nucleic acids of interest (e.g.,
genomic or mitochondrial DNA or messenger RNA (mRNA)) from an
organism. Then it is necessary to isolate the nucleic acids of
interest from the mixture of all nucleic acids and usually, to
amplify these nucleic acids to obtain quantities suitable for their
characterisation and/or detection. Isolating the nucleic fragments
has been considered necessary even when one is interested in a
representative but random set of all of the different nucleic
acids, for instance, a representative set of all the mRNAs present
in a cell or of all the fragments obtained after genomic DNA has
been cut randomly into small pieces.
Several methods can be used to amplify DNA with biological means
and are well known by those skilled in the art. Generally, the
fragments of DNA are first inserted into vectors with the use of
restriction enzymes and DNA ligases. A vector containing a fragment
of interest can then be introduced into a biological host and
amplified by means of well established protocols. Usually hosts are
randomly spread over a growth medium (e.g. agar plates). They can
then replicate to provide colonies that originated from individual
host cells.
Up to millions of simultaneous amplification of cloned DNA
fragments can be carried out simultaneously in such hosts. The
density of colonies is of the order of 1 colony/mm2. In order to
obtain DNA from such colonies one option is to transfer the
colonies to a membrane, and then to immobilise the DNA from within
the biological hosts directly to the membrane (Grunstein, M. and D.
S. Hogness, Colony Hybridization: A method for the isolation of
cloned DNAs that contain a specific gene, Proceedings of the
National Academy of Science, USA, 72:3961 (1975)). With these
options however, the amount of transferred DNA is limited and often
insufficient for non-radioactive detection.
Another option is to transfer by sterile technique individually
each colony into a container (e.g., 96 well plates) where further
host cell replication can occur so that more DNA can be obtained
from the colonies. Amplified nucleic acids can be recovered from
the host cells with an appropriate purification process. However
such a procedure is generally time and labor consuming, and
difficult to automate.
The revolutionary technique of DNA amplification using the
polymerase chain reaction (PCR) was proposed in 1985 by Mullis et
al. (Saiki, R., S. Scharf, F. Faloona, K. Mullis, G. Horn, H.
Erlich and N. Arnheim, Science 230, 1350-1354 (1985) and is now
well known by those skilled in the art. In this amplification
process, a DNA fragment of interest can be amplified using two
short (typically about 20 base long) oligonucleotides that flank a
region to be amplified, and that are usually referred to as
"primers". Amplification occurs during the PCR cycling, which
includes a step during which double stranded DNA molecules are
denatured (typically a reaction mix is heated, e.g. to 95.degree.
C. in order to separate double stranded DNA molecules into two
single stranded fragments), an annealing step (where the reaction
mix is brought to e.g., 45.degree. C. in order to allow the primers
to anneal to the single stranded templates) and an elongation step
(DNA complementary to the single stranded fragment is synthesised
via sequential nucleotide incorporation at the ends of the primers
with the DNA polymerase enzyme).
The above procedure is usually performed in solution, whereby
neither the primers nor a template are linked to any solid
matrix.
More recently, however, it has been proposed to use one primer
grafted to a surface in conjunction with free primers in solution
in order to simultaneously amplify and graft a PCR product onto the
surface (Oroskar, A. A., S. E. Rasmussen, H. N. Rasmussen, S. R.
Rasmussen, B. M. Sullivan, and A. Johansson, Detection of
immobilised amplicons by ELISA-like techniques, Clinical Chemistry
42:1547 (1996)). (The term "graft" is used herein to indicate that
a moiety becomes attached to a surface and remains there unless and
until it is desired to remove it.) The amplification is generally
performed in containers (e.g., in 96 well format plates) in such a
way that each container contains the PCR product(s) of one
reaction. With such methods, some of the PCR product becomes
grafted to a surface of the container having primers therein which
has been in contact with the reactant during the PCR cycling. The
grafting to the surface simplifies subsequent assays and allows
efficient automation.
Arraying of DNA samples is more classically performed on membranes
(e.g., nylon or nitro-cellulose membranes). With the use of
suitable robotics (e.g., Q-bot.TM., Genetix ltd, Dorset BH23 3TG
UK) it is possible to reach a density of up to 10 samples/mm.sup.2.
Here, the DNA is covalently linked to a membrane by physicochemical
means (e.g., UV irradiation). These technologies allow the arraying
of large DNA molecules (e.g. molecules over 100 nucleotides long)
as well as smaller DNA molecules. Thus both templates and probes
can be arrayed.
New approaches based on pre-arrayed glass slides (arrays of
reactive areas obtained by ink-jet technology (Blanchard, A. P. and
L. Hood, Oligonucleotide array synthesis using ink jets, Microbial
and Comparative Genomics, 1:225 (1996)) or arrays of reactive
polyacrylamide gels (Yershov, G. et al., DNA analysis and
diagnostics on oligonucleotide microchips, Proceedings of the
National Academy of Science, USA, 93:4913-4918 (1996)) allow the
arraying of up to 100 samples/mm.sup.2. With these technologies,
only probe (oligonucleotide) grafting has been reported. Reported
number of samples/mm.sup.2 are still fairly low (25 to 64).
Higher sample densities are achievable by the use of DNA chips,
which can be arrays of oligonucleotides covalently bound to a
surface and can be obtained with the use of micro-lithographic
techniques (Fodor, S. P. A. et al., Light directed, spatially
addressable parallel chemical synthesis, Science 251:767(1991)).
Currently, chips with 625 probes/mm.sup.2 are used in applications
for molecular (Lockhart, D. J. et al., Expression monitoring by
hybridisation to high-density oligonucleotide arrays, Nature
Biotechnology 14:1675 (1996)). Probe densities of up to 250 000
samples/cm.sup.2 are claimed to be achievable (Chee, M. et al.,
Accessing genetic information with high-density DNA arrays, Science
274:610 (1996)). Currently, up to 132000 different oligonucleotides
can be arrayed on a single chip of approximately 2.5 cm.sup.2.
Presently, these chips are manufactured by direct solid phase
oligonucleotide synthesis with the 3' OH end of the oligo attached
to the surface. Thus these chips have been used to provide
oligonucleotide probes which cannot act as primers in a DNA
polymerase-mediated elongation step.
When PCR products are linked to the vessel in which PCR
amplification takes place, this can be considered as a direct
arraying process. The density of the resultant array of PCR
products is then limited by the available vessel. Currently
available vessels are only in 96 well microtiter plate formats.
These allow only around .about.0.02 samples of PCR
products/mm.sup.2 of surface to be obtained.
Using the commercially available Nucleolink.TM. system obtainable
from Nunc A/S (Roskilde, Denmark) it is possible to achieve
simultaneous amplification and arraying of samples in containers on
the surface of which oligonucleotide primers have been grafted.
However, in this case the density of the array of samples is fixed
by the size of the vessel. Presently a density of 0.02
samples/mm.sup.2 is achievable for the 96 well plate format.
Increasing this density is difficult. This is apparent since, for
instance, the availability of 384 well plates (0.08
samples/mm.sup.2) suitable for PCR has been delayed due to
technical problems (e.g. heat transfer and capillary effects during
filling). It is thus unlikely that orders of magnitude improvements
in the density of samples arrayed with this approach can be
achieved in the foreseeable future.
The present invention aims to overcome or at least alleviate some
of the disadvantages of prior art methods of nucleic acid
amplification.
According to the present invention there is provided a method of
nucleic acid amplification, comprising the steps of: A. providing a
plurality of primers that are immobilised but that have one end
exposed to allow primer extension; B. allowing a single stranded
target nucleic acid molecule to anneal to one of said plurality of
primers over part of the length of said single stranded nucleic
acid molecule and then extending that primer using the annealed
single stranded nucleic acid molecule as a template, so as to
provide an extended immobilised nucleic acid strand; C. separating
the target nucleic molecule from the extended immobilised nucleic
acid strand; D. allowing the extended immobilised nucleic acid
strand to anneal to one of said plurality of primers referred to in
step A) and then extending that primer using the extended
immobilized nucleic acid strand as a template, so as to provide
another extended immobilised nucleic acid strand; and optionally,
E. separating the annealed extended immobilised nucleic acid
strands from one another.
Preferably the method also comprises the step of: F. using at least
one extended immobilised nucleic acid strand to repeat steps D) and
E), so as to provide additional extended immobilised nucleic acid
strands and, optionally, G. repeating step F) one or more
times.
Desirably the single-stranded target nucleic acid sequence is
provided by a method in which said single-stranded target nucleic
acid is produced by providing a given nucleic acid sequence to be
amplified (which sequence may be known or unknown) and adding
thereto a first nucleic acid sequence and a second nucleic acid
sequence; wherein said first nucleic acid sequence hybridises to
one of said plurality of primers and said second nucleic acid
sequence is complementary to a sequence which hybridises to one of
said plurality of primers.
The second nucleic acid sequence may be a sequence that is the same
as the sequence of one of the plurality of primers. Thus the
single-stranded target nucleic acid sequence may be provided by a
method in which said single-stranded target nucleic acid is
produced by providing a given nucleic acid sequence to be amplified
(which sequence may be known or unknown) and adding thereto a first
nucleic acid sequence and a second nucleic acid sequence; wherein
said first nucleic acid sequence hybridises to one of said
plurality of primers and said second nucleic acid sequence is the
same as the sequence of one of said plurality of primers.
The first and second nucleic acid sequences may be provided at
first and second ends of said single-stranded target nucleic acid,
although this is not essential.
If desired a tag may be provided to enable amplification products
of a given nucleic acid sequence to be identified.
Colonies
The method of the present invention allows one or more distinct
areas to be provided, each distinct area comprising a plurality of
immobilised nucleic acid strands (hereafter called "colonies").
These areas can contain large numbers of amplified nucleic acid
molecules. These molecules may be DNA and/or RNA molecules and may
be provided in single or double stranded form. Both a given strand
and its complementary strand can be provided in amplified form in a
single colony.
Colonies of any particular size can be provided.
However, preferred colonies measure from 10 nm to 100 .mu.m across
their longest dimension, more preferably from 100 nm to 10 .mu.m
across their longest dimension. Desirably a majority of the
colonies present on a surface (i.e. at least 50% thereof) have
sizes within the ranges given above.
Colonies can be arranged in a predetermined manner or can be
randomly arranged. Two or three dimensional colony configurations
are possible. The configurations may be regular (e.g. having a
polygonal outline or having a generally circular outline) or they
may be irregular.
Colonies can be provided at high densities. Densities of over one
colony/mm.sup.2 of surface can be achieved. Indeed densities of
over 10.sup.2, over 10.sup.3 or even over 10.sup.4
colonies/mm.sup.2 are achievable using the present invention. In
preferred embodiments, the present invention provides colony
densities of 10.sup.4-5 colonies/mm.sup.2, more preferably
densities of 10.sup.6-7 colonies/mm.sup.2, thus offering an
improvement of 3 to 4 orders of magnitude relative to densities
achievable using many of the prior art methods. It is this property
of the invention that allows a great advantage over prior art,
since the high density of DNA colonies allows a large number of
diverse DNA templates (up to 10.sup.6-7 colonies/mm.sup.2, to be
randomly arrayed and amplified.
Primers
The immobilised primers for use in the present invention can be
provided by any suitable means, as long as a free 3'-OH end is
available for primer extension. Where many different nucleic acid
molecules are to be amplified, many different primers may be
provided. Alternatively "universal" primers may be used, whereby
only one or two different types of primer (depending upon the
embodiment of the invention) can be used to amplify the different
nucleic acid molecules. Universal primers can be used where the
molecules to be amplified comprise first and second sequences, as
described previously. The provision of universal primers is
advantageous over methods such as those disclosed in W096/04404
(Mosaic Technologies, Inc.) where specific primers must be prepared
for each particular sequence to be amplified.
Synthetic oligodeoxynucleotide primers are available commercially
from many suppliers (e.g. Microsynth, Switzerland, Eurogentech,
Belgium).
Grafting of primers onto silanized glass or quartz and grafting of
primers onto silicon wafers or gold surface has been described
(Maskos, U. and E. M Southern, Oligonucleotide hybridizations on
glass supports: a novel linker for oligonucleotide synthesis and
hybridization properties of oligonucleotides synthesised in situ,
Nucleic Acids Research 20(7):1679-84, 1992; Lamture, J. B., et. al.
Direct-detection of nucleic-acid hybridization on the surface of a
charge-coupled-device, Nucleic Acids Research 22(11):2121-2125,
1994; Chrisey, L. A., G. U. Lee, and C. E. Oferrall, Covalent
attachment of synthetic DNA to self-assembled monolayer films,
Nucleic Acids Research 24(15):3031-3039, 1996).
Grafting biotinylated primers to supports covered with streptavidin
is another alternative. This grafting method is commonly used for
bio-macromolecules in general.
Non covalent grafting of primers at the interface between an
aqueous phase and a hydrophobic phase through a hydrophobic anchor
is also possible for the present invention. Such anchoring is
commonly used for bio-macromolecules in general (S. Terrettaz et
al.: Protein binding to supported lipid membranes, Langmuir 9, 1361
(1993)). Preferred forms of such interfaces would be liposomes,
lipidic vesicles, emulsions, patterned bilayers, Langmuir or
Langmuir-Blodgett films. The patterns may be obtained by directed
pattering on templates, e.g., silicon chips patterned through
micro-lithographic methods (Goves, J. T. et al., Micropatterning
Fluid Bilayers on Solid Supports, in Science 275, 651 (1997)). The
patterns may also be obtained by due to the self-assembly
properties of "colloids", e.g., emulsions or latex particles
(Larsen, A. E. and D. G. Grier, Like charge attractions in
metastable colloidal crystallites, Nature 385,230 (1997)).
In the above methods, one, two or more different primers can be
grafted onto a surface. The primers can be grafted homogeneously
and simultaneously over the surface.
Using microlithographic methods it is possible to provide
immobilised primers in a controlled manner. If direct synthesis of
oligonucleotides onto a solid support with a free 3'-OH end is
desired, then micro-lithographic methods can be used to
simultaneously synthesise many different oligonucleotide primers
(Pirrung, M. C. and Bradley, J. C. Comparison of methods for
photochemical phosphoramidite-based DNA-synthesis. Journal Of
Organic Chemistry 60(20):6270-6276, 1995). These may be provided in
distinct areas that may correspond in configuration to colonies to
be formed, (e.g. they may be several nanometers or micrometers
across). Within each area, only a single type of primer
oligonucleotide need be provided. Alternatively a mixture
comprising a plurality of different primers may be provided. In
either case, primers can be homogeneously distributed within each
area. They may be provided in the form of a regular array.
Where areas initially comprise only one type of immobilised primer
they may be modified, if desired, to carry two or more different
types of primer. One way to achieve this is to use molecules as
templates for primer extension that have 3' ends that hybridise
with a single type of primer initially present and that have 5'
ends extending beyond the 3' ends of said primers. By providing a
mixture of templates with different sequences from one another,
primer extension of one type of primer using the mixture of such
templates followed by strand separation will result in different
modified primers. (The modified primers are referred to herein as
"extended" primers in order to distinguish from the "primary"
primers initially present on a surface).
One, two or more different types of extended primer can be provided
in this manner at any area where primary primers are initially
located. Substantially equal portions of different templates can be
used, if desired, in order to provide substantially equal
proportions of different types of immobilised extended primer over
a given area. If different proportions of different immobilised
extended primers are desired, then this can be achieved by
adjusting the proportions of different template molecules initially
used accordingly.
A restriction endonuclease cleavage site may be located within the
primer. A primer may also be provided with a restriction
endonuclease recognition site which directs DNA cleavage several
bases distant (Type II restriction endonucleases). (For the
avoidance of doubt, such sites are deemed to be present even if the
primer and its complement are required to be present in a double
stranded molecule for recognition and/or cleavage to occur.)
Alternatively a cleavage site and/or a recognition site may be
produced when a primer is extended. In any event, restriction
endonucleases can be useful in allowing an immobilised nucleic acid
molecule within a colony to be cleaved so as to release at least a
part thereof. As an alternative to using other restriction
endonucleases, ribozymes can be used to release at least parts of
nucleic acid molecules from a surface (when such molecules are RNA
molecules). Other methods are possible. For example if a covalent
bond is used to link a primer to a surface this bond may be broken
(e.g. by chemical, physical or enzymatic means).
Primers for use in the present invention are preferably at least
five bases long. Normally they will be less than 100 or less than
50 bases long. However this is not essential. Naturally occurring
and/or non-naturally occurring bases may be present in the
primers.
Target Nucleic Acid Molecules
Turning now to target nucleic acid molecules (also referred to
herein as "templates") for use in the method of the present
invention, these can be provided by any appropriate means. A target
molecule (when in single-stranded form) comprises a first part
having a sequence that can anneal with a first primer and a second
part having a sequence complementary to a sequence that can anneal
with a second primer. In a preferred embodiment the second part has
the same sequence as the second primer.
The second primer may have a sequence that is the same as, or
different from, the sequence of the first primer.
The first and second parts of the target nucleic acid molecules are
preferably located at the 3' and at the 5' ends respectively
thereof. However this is not essential. The target molecule will
usually also comprise a third part located between the first and
second parts. This part of the molecule comprises a particular
sequence to be replicated. It can be from any desired source and
may have a known or unknown (sometimes referred to as "anonymous")
sequence. It may be derived from random fractionation by mechanical
means or by limited restriction enzyme digestion of a nucleic acid
sample, for example.
Further parts of the target molecules may be provided if desired.
For example parts designed to act as tags may be provided. A "tag"
is defined by its function of enabling a particular nucleic acid
molecule (or its complement) to be identified.
Whatever parts are present, target nucleic acid molecules can be
provided by techniques known to those skilled in the art of nucleic
acid manipulation. For example, two or more parts can be joined
together by ligation. If necessary, prior to ligation appropriate
modifications can be made to provide molecules in a form ready for
ligation. For example if blunt end ligation is desired then a
single-strand specific exonuclease such as S1 nuclease could be
used to remove single stranded portions of molecules prior to
ligation. Linkers and/or adapters may also be used in nucleic acid
manipulation. (Techniques useful for nucleic acid manipulation are
disclosed in Sambrook et al, Molecular Cloning, 2nd Edition, Cold
Spring Harbor Laboratory Press (1989), for example.)
Once a template molecule has been synthesised it can be cloned into
a vector and can be amplified in a suitable host before being used
in the present invention. It may alternatively be amplified by PCR.
As a further alternative, batches of template molecules can be
synthesised chemically using automated DNA synthesisers (e.g. from
Perkin-Elmer/Applied Biosystems, Foster City, Calif.).
It is however important to note that the present invention allows
large numbers of nucleic acid molecules identical in sequence to be
provided in a colony arising from a single molecule of template.
Furthermore, the template can be re-used to generate further
colonies. Thus it is not essential to provide large numbers of
template molecules to be used in colony formation.
The template can be of any desired length provided that it can
participate in the method of the present invention. Preferably it
is at least 10, more preferably at least 20 bases long. More
preferably it is at least 100 or at least 1000 bases long. As is
the case for primers for use in the present invention, templates
may comprise naturally occurring and/or non-naturally occurring
bases.
Reaction Conditions
Turning now to reaction conditions suitable for the method of the
present invention, it will be appreciated that the present
invention uses repeated steps of annealing of primers to templates,
primer extension and separation of extended primers from templates.
These steps can generally be performed using reagents and
conditions known to those skilled in PCR (or reverse transcriptase
plus PCR) techniques. PCR techniques are disclosed, for example, in
"PCR: Clinical Diagnostics and Research", published in 1992 by
Springer Verlag.
Thus a nucleic acid polymerase can be used together with a supply
of nucleoside triphosphate molecules (or other molecules that
function as precursors of nucleotides present in DNA/RNA, such as
modified nucleoside triphosphates) to extend primers in the
presence of a suitable template.
Excess deoxyribonucleoside triphosphates are desirably provided.
Preferred deoxyribonucleoside triphosphates are abbreviated; dTTP
(deoxythymidine nucleoside triphosphate), dATP (deoxyadenosine
nucleoside triphosphate), dCTP (deoxycytosine nucleoside
triphosphate) and dGTP (deoxyguanosine nucleoside triphosphate).
Preferred ribonucleoside triphosphates are UTP, ATP, CTP and GTP.
However alternatives are possible. These may be naturally or
nonnaturally occurring. A buffer of the type generally used in PCR
reactions may also be provided.
A nucleic acid polymerase used to incorporate nucleotides during
primer extension is preferably stable under the pertaining reaction
conditions in order that it can be used several times. (This is
particularly useful in automated amplification procedures.) Thus,
where heating is used to separate a newly synthesised nucleic acid
strand from its template, the nucleic acid polymerase is preferably
heat stable at the temperature used. Such heat stable polymerases
are known to those skilled in the art. They are obtainable from
thermophilic micro-organisms. They include the DNA dependent DNA
polymerase known as Taq polymerase and also thermostable
derivatives thereof. (The nucleic acid polymerase need not however
be DNA dependent. It may be RNA dependent. Thus it may be a reverse
transcriptase--i.e. an RNA dependent DNA polymerase).
Typically, annealing of a primer to its template takes place at a
temperature of 25 to 90.degree. C. Such a temperature range will
normally be maintained during primer extension. Once sufficient
time has elapsed to allow annealing and also to allow a desired
degree of primer extension to occur, the temperature can be
increased, if desired, to allow strand separation. At this stage
the temperature will typically be increased to a temperature of 60
to 100.degree. C. [High temperatures can also be used to reduce
non-specific priming problems prior to annealing. They can be used
to control the timing of colony initiation, e.g. in order to
synchronise colony initiation for a number of samples.]
Alternatively, the strands maybe separated by treatment with a
solution of low salt and high pH (>12) or by using a chaotropic
salt (e.g. guanidinium hydrochloride) or by an organic solvent
(e.g. formamide).
Following strand separation (e.g. by heating), preferably a washing
step will be performed. The washing step can be omitted between
initial rounds of annealing, primer extension and strand
separation, if it is desired to maintain the same templates in the
vicinity of immobilised primers. This allows templates to be used
several times to initiate colony formation. (It is preferable to
provide a high concentration of template molecules initially so
that many colonies are initiated at one stage).
The size of colonies can be controlled, e.g. by controlling the
number of cycles of annealing, primer extension and strand
separation that occur. Other factors which affect the size of
colonies can also be controlled. These include the number and
arrangement on a surface of immobilised primers, the conformation
of a support onto which the primers are immobilised, the length and
stiffness of template and/or primer molecules, temperature and the
ionic strength and viscosity of a fluid in which the
above-mentioned cycles can be performed.
Uses of Colonies
Once colonies have been formed they can be used for any desired
purpose.
For example, they may be used in nucleic acid sequencing (whether
partial or full), in diagnosis, in screening, as supports for other
components and/or for research purposes (preferred uses will be
described in greater detail later on). If desired colonies may be
modified to provide different colonies (referred to herein as
"secondary colonies" in order to distinguish from the "primary
colonies" initially formed).
Surfaces Comprising Immobilised Nucleic Acid Strands
A surface comprising immobilised nucleic acid strands in the form
of colonies of single stranded nucleic acid molecules is also
within the scope of the present invention.
Normally each immobilised nucleic acid strand within a colony will
be located on the surface so that an immobilised and complementary
nucleic acid strand thereto is located on the surface within a
distance of the length of said immobilised nucleic acid strand
(i.e. within the length of one molecule). This allows very high
densities of nucleic acid strands and their complements to be
provided in immobilised form. Preferably there will be
substantially equal proportions of a given nucleic acid strand and
its complement within a colony. A nucleic acid strand and its
complement will preferably be substantially homogeneously
distributed within the colony.
It is also possible to provide a surface comprising single stranded
nucleic acid strands in the form of colonies, where in each colony,
the sense and anti-sense single strands are provided in a form such
that the two strands are no longer at all complementary, or simply
partially complementary. Such surfaces are also within the scope of
the present invention. Normally, such surfaces are obtained after
treating primary colonies, e.g., by partial digestion by
restriction enzymes or by partial digestion after strand separation
(e.g., after heating) by an enzyme which digests single stranded
DNA), or by chemical or physical means, (e.g., by irradiating with
light colonies which have been stained by an intercalating dye
e.g., ethidium bromide).
Once single stranded colonies have been provided they can be used
to provide double stranded molecules. This can be done, for
example, by providing a suitable primer (preferably in solution)
that hybridises to the 3' ends of single stranded immobilised
molecules and then extending that primer using a nucleic acid
polymerase and a supply of nucleoside triphosphates (or other
nucleotide precursors).
Thus surfaces comprising colonies of non-bridged double stranded
nucleic acid molecules are also within the scope of the present
invention. (The term "non-bridged" is used here to indicate that
the molecules are not in the form of the bridge-like structures
shown in e.g. FIG. 1h.)
Using the present invention, small colonies can be provided that
contain large numbers of nucleic acid molecules (whether single or
double stranded). Many colonies can therefore be located on a
surface having a small area. Colony densities that can be obtained
may therefore be very high, as discussed supra.
Different colonies will generally be comprised of different
amplified nucleic acid strands and amplified complementary strands
thereto. Thus the present invention allows many different
populations of amplified nucleic acid molecules and their
complements to be located on a single surface having a relatively
small surface area. The surface will usually be planar, although
this is not essential.
Apparatuses
The present invention also provides an apparatus for providing a
surface comprising colonies of the immobilised nucleic acid
molecules discussed supra.
Such an apparatus can include one or more of the following: a)
means for immobilising primers on a surface (although this is not
needed if immobilised primers are already provided); b) a supply of
a nucleic acid polymerase; c) a supply of precursors of the
nucleotides to be incorporated into a nucleic acid (e.g. a supply
of nucleoside triphosphates); d) means for separating annealed
nucleic acids (e.g. heating means); and e) control means for
co-ordinating the different steps required for the method of the
present invention.
Other apparatuses are within the scope of the present invention.
These allow immobilised nucleic acids produced via the method of
the present invention to be analysed. They can include a source of
reactants and detecting means for detecting a signal that may be
generated once one or more reactants have been applied to the
immobilised nucleic acid molecules. They may also be provided with
a surface comprising immobilised nucleic acid molecules in the form
of colonies, as described supra.
Desirably the means for detecting a signal has sufficient
resolution to enable it to distinguish between signals generated
from different colonies.
Apparatuses of the present invention (of whatever nature) are
preferably provided in automated form so that once they are
activated, individual process steps can be repeated
automatically.
The present invention will now be described without limitation
thereof in sections A to I below with reference to the accompanying
drawings.
It should be appreciated that procedures using DNA molecules
referred to in these sections are applicable mutatis mutandis to
RNA molecules, unless the context indicates otherwise.
It should also be appreciated that where sequences are provided in
the following description, these are written from 5' to 3' (going
from left to right), unless the context indicates otherwise.
The figures provided are summarised below:
FIG. 1A and FIG. 1B illustrate a method for the simultaneous
amplification and immobilisation of nucleic acid molecules using a
single type of primer.
FIG. 2 illustrates how colony growth using a method of the present
invention can occur.
FIG. 3 illustrates the principle of the method used to produce DNA
colonies using the present invention. It also illustrates the
annealing, elongation and denaturing steps that are used to provide
such colonies.
FIG. 4 is an example of DNA colonies formed by amplification of a
specific template with single primers grafted onto a surface.
FIG. 5 is an example of DNA colonies formed by amplification of a
specific template with single primers grafted onto a surface.
FIGS. 6A and FIG. 6B illustrate a method for the simultaneous
amplification and immobilization of nucleic acid molecules using
two types of primer.
FIG. 7 shows actual DNA colonies produced via the present
invention.
FIG. 8 shows actual DNA colonies produced via the present
invention.
FIG. 9A and FIG. 9B illustrate a method of the simultaneous
amplification and immobilization of nucleic acid molecules when a
target molecule is used as a template having internal sequences
that anneal with primers.
FIG. 10A and FIG. 10B illustrate a method to synthesise additional
copies of the original nucleic acid strands using nucleic acid
strands present in colonies. The newly synthesised strands are
shown in solution but can be provided in immobilised form if
desired.
FIG. 11A and FIG. 11B show the PCR amplification of DNA from DNA
found in the pre-formed DNA colonies.
FIG. 12A, FIG. 12B, and FIG. 12C illustrate how secondary primers
can be generated from DNA colonies.
FIG. 13A and FIG. 13B illustrate how secondary DNA colonies can be
generated from secondary primers.
FIG. 14A and FIG. 14B illustrate how primers with different
sequences can be generated from a surface functionalised with
existing primers.
FIG. 15 depicts methods of preparing DNA fragments suitable for
generating DNA colonies.
FIG. 16 illustrates a method for synthesising cRNA using the DNA
colony as a substrate for RNA polymerase.
FIG. 17 illustrates a preferable method to determine the DNA
sequence of DNA present in individual colonies.
FIG. 18 illustrates a method of determining the sequence of a DNA
colony, de novo.
FIG. 19 illustrates the utility of secondary DNA colonies in the
assay of mRNA expression levels.
FIGS. 20 and 21 illustrates the use of the secondary DNA colonies
in the isolation and identification of novel and rare expressed
genes.
A) SCHEME SHOWING THE SIMULTANEOUS AMPLIFICATION AND IMMOBILISATION
OF NUCLEIC ACID MOLECULES USING A SINGLE TYPE OF PRIMER
Referring now to FIG. 1A(a), a surface is provided having attached
thereto a plurality of primers (only one primer is shown for
simplicity). Each primer (1) is attached to the surface by a
linkage indicated by a dark block. This may be a covalent or a
non-covalent linkage but should be sufficiently strong to keep a
primer in place on the surface. The primers are shown having a
short nucleotide sequence (5'-ATT). In practice however longer
sequences would generally be provided.
FIG. 1A(b) shows a target molecule (II) that has annealed to a
primer. The target molecule comprises at its 3' end a sequence (5'
ATT) that is complementary to the primer sequence (5'-ATT). At its
5' end the target molecule comprises a sequence (5'-ATT) that is
the same as the primer sequence (although exact identity is not
required).
Between the two ends any sequence to be amplified (or the
complement of any sequence to be amplified) can be provided. By way
of example, part of the sequence to be amplified has been shown as
5'-CCG.
In FIG. 1A(c) primer extension is shown. Here a DNA polymerase is
used together with dATP, dTTP, dGTP and dCTP to extend the primer
(5'-ATT) from its 3' end, using the target molecule as a
template.
When primer extension is complete, as shown in FIG. 1A(d), it can
be seen that an extended immobilised strand (III) is provided that
is complementary to the target molecule. The target molecule can
then be separated from the extended immobilised strand (e.g. by
heating, as shown in FIG. 1A(e)). This separation step frees the
extended, immobilised strand so that it can then be used to
initiate a subsequent round of primer extension, as shown in FIGS.
1A(f) and 1A(g). Here the extended, immobilised strand bends over
so that one end of that strand (having the terminal sequence
5'-AAT) anneals with another primer (2,5'-ATT), as shown in FIG.
1A(f). That primer provides a 3' end from which primer extension
can occur, this time using the extended, immobilised strand as a
template. Primer extension is shown occurring in FIG. 1A(g) and is
shown completed in FIG. 1A(h).
FIG. 1B(i) shows the two extended immobilised strands that were
shown in FIG. 1A(h) after separation from one another (e.g. by
heating). Each of these strands can then themselves be used as
templates in further rounds of primer extension initiated from new
primers (3 and 4), as shown in FIGS. 1B(j) and 1B(k). Four single
stranded, immobilised strands can be provided after two rounds of
amplification followed by a strand separation step (e.g. by
heating), as shown in FIG. 1B(l). Two of these have sequences
corresponding to the sequence of the target molecule originally
used as a template. The other two have sequences complementary to
the sequence of the target molecule originally used as a template.
(In practice a given immobilised strand and its immobilised
complement may anneal once).
It will therefore be appreciated that a given sequence and its
complement can be provided in equal numbers in immobilised form and
can be substantially homogeneously distributed within a colony.
Further rounds of amplification beyond those shown in FIG. 1 can of
course be performed so that colonies comprising large numbers of a
given single stranded nucleic acid molecule and a complementary
strand thereto can be provided. Only a single template need be used
to initiate each colony, although, if desired, a template can be
reused to initiate several colonies.
It will be appreciated that the present invention allows very high
densities of immobilised extended nucleic acid molecules to be
provided. Within a colony each extended immobilised molecule will
be located at a surface within one molecule length of another
extended immobilised molecule. Thus position 3 shown in FIG. 1B(l)
is within one molecule length of position 1; position 1 is within
one molecule length of position 2; and position 2 is within one
molecule length of position 4.
FIG. 2 is provided to illustrate how colony growth can occur (using
the method described with reference to FIG. 1 and to FIG. 6 or any
other method of the present invention for providing immobilised
nucleic acid molecules).
A flat plate is shown schematically in plan view having primers
immobilised thereon in a square grid pattern (the primers are
indicated by small dots). A regular grid is used solely for
simplicity: in many real cases, the positions of the primers might
indeed be less ordered or random.
At the position indicated by arrow X a template molecule has
annealed to a primer and an initial bout of primer extension has
occurred to provide an immobilised, extended nucleic acid strand.
Following strand separation, an end of that strand becomes free to
anneal to further primers so that additional immobilised, extended
nucleic acid strands can be produced. This is shown having occurred
sequentially at positions indicated by the letter Y. For
simplicity, the primer chosen for annealing is positioned next to
the primer carrying the nucleic acid strand: in real cases, the
nucleic acid strand could anneal with a primer which is not its
next nearest neighbor. However, this primer will obviously be
within a distance equal to the length of the nucleic acid
strand.
It will be appreciated that annealing at only one (rather than at
all) of these positions is required for colony cell growth to
occur.
After immobilised, extended, single-stranded nucleic acid molecules
have been provided at the positions indicated by letter Y, the
resultant molecules can themselves anneal to other primers and the
process can be continued to provide a colony comprising a large
number of immobilised nucleic acid molecules in a relatively small
area.
FIG. 3 shows a simplified version of the annealing, elongation and
denaturing cycle. It also depicts the typical observations that can
be made, as can be seen on the examples shown in FIGS. 4 and 6. The
simultaneous amplification and immobilisation of nucleic acids
using solid phase primers has been successfully achieved using the
procedure described in Examples 1, 2 and 3 below:
Example 1
Oligonucleotides, phosphorylated at their 5'-termini (Microsynth
GmbH, Switzerland), were grafted onto Nucleolink plastic microtitre
wells (Nunc, Roskilde, Denmark). The sequence of the
oligonucleotide p57 corresponds to the sequence
5'-TTTTTCACCAACCCAAACCAACCCAAACC (SEQ ID NO:1) and p58 corresponds
to the sequence 5'-TTTTTTAGAAGGAGAAGGAAAGGGAAAGGG (SEQ ID NO:2).
Microtitre wells with p57 or p58 were prepared as follows. In each
Nucleolink well, 30 .mu.l of a 160 nM solution of the
oligonucleotide in 10 mM 1-methyl-imidazole (pH 7.0) (Sigma
Chemicals, St. Louis, Mo.) was added. To each well, 10 .mu.l of 40
mM 1-ethyl-3-(3dimethylaminopropyl)-carbodiimide (pH 7.0) (Sigma
Chemicals) in 10 mM 1-methyl-imidazole, was added to the solution
of oligonucleotides. The wells were then sealed and incubated at
50.degree. C. overnight. After the incubation, wells were rinsed
twice with 200 .mu.l of RS (0.4N NaOH, 0.25% Tween 20 (Fluka
Chemicals, Switzerland)), incubated 15 minutes with 200 .mu.l RS,
washed twice with 200 .mu.l RS and twice with 200 .mu.l TNT (100 mM
TrisHCl pH7.5, 150 mM NaCl, 0.1% Tween 20). Tubes were dried at
50.degree. C. and were stored in a sealed plastic bag at 4.degree.
C.
Colony generation was initiated in each well with 15 .mu.l of
priming mix; 1 nanogram template DNA (where the template DNA began
with the sequence 5'-AGAAGGAGAAGGAAAGGGAAAGGG (SEQ ID NO:3) and
terminated at the 3'-end with the sequence
CCCTTTCCCTTTCCTTCTCCTTCT-3') (SEQ ID NO:4), the four dNTPs (0.2
mM), 0.1% BSA (bovine serum albumin, Boehringer-Mannheim, Germany),
0.1% Tween 20, 8% DMSO (dimethylsulfoxide, Fluka Chemicals,
Switzerland), 1.times. Amplitaq PCR buffer and 0.025 units/.mu.l of
AmpliTaq DNA polymerase (Perkin Elmer, Foster City, Calif.). The
priming reaction was a single round of PCR under the following
conditions; 94.degree. C. for 4 minutes, 60.degree. C. for 30
seconds and 72.degree. C. for 45 seconds in a thermocycler (PTC
200, MJ Research, Watertown, Mass.). Then 100 .mu.l TE buffer (10
mM trisHCl, pH 7.5, 1 mM EDTA) was used in three successive one
minute long washes at 94.degree. C. The DNA colonies were then
formed by adding to each well, 20 .mu.l of polymerisation mix,
which was identical to the priming mix but lacking the template
DNA. The wells were then placed in the PTC 200 thermocycler and
colony growing was performed by incubating the sealed wells 4
minutes at 94.degree. C. and cycling for 50 repetitions the
following conditions: 94.degree. C. for 45 seconds, 65.degree. C.
for 2 minutes, 72.degree. C. for 45 seconds. After completion of
this program, the wells were kept at 8.degree. C. until further
use.
A 640 base pair fragment corresponding to the central sequence of
the template (but not including the 5'-AGAAGGAGAAGGAAAGGGAAAGGG
(SEQ ID NO:3) sequence) was amplified by PCR. The isolated fragment
was labeled with biotin N.sup.4-dCTP (NEN Life Sciences, Boston,
Mass.) and a trace of [.alpha.-.sup.32P]dCTP (Amersham, UK) using
the Prime-it II labeling kit (Stratagene, San Diego, Calif.) to
generate a biotinylated probe.
The biotinylated probe was diluted in to a concentration of 2.5 nM
in EasyHyb (Boehringer-Mannheim, Germany) and 15 .mu.l was
hybridized to each sample with the following temperature scheme
(PTC 200 thermocycler): 94.degree. C. for 5 minutes, followed by
500 steps of 0.1.degree. C. decrease in temperature every 12
seconds (in other words, the temperature is decreased down to
45.degree. C. in 100 minutes). The samples are then washed as
follows; 1 time with 2.times.SSC/0.1% SDS (2.times.SSC; 0.3M
NaCl/0.03M sodium citrate pH7.0/0.001 mg/ml sodium dodecyl sulfate)
at room temperature, once with 2.times.SSC/0.1% SDS at 37.degree.
C. and once with 0.2.times.SSC/0.1% SDS at 50.degree. C. The wells
are then incubated for 30 minutes with 50 .mu.l of red fluorescent,
Neutravidin-coated, 40 nm FluoSpheres.RTM. (580 nm excitation and
605 nm emission, Molecular Probes Inc., Eugene, Oreg.) in TNT/0.1%
BSA. (The solution of microspheres is made from a dilution of 2
.mu.l of the stock solution of microspheres into 1 ml of TNT/0.1%
BSA, which is then sonicated for 5 minutes in a 50 W ultra-sound
water-bath (Elgasonic, Switzerland), followed by filtration through
a 0.22 .mu.m filter (Millex GV4). The wells are then counted
(Cherenkov) on a Microbeta plate scintillation counter (WALLAC,
Turku, Finland).
ExcessFluoSpheres.RTM. are removed by washing for 30 min in
TNT/0.1% BSA at room temperature. Images of the stained samples are
observed using a 20.times. objective on an inverted microscope
(Axiovert S100TV, Carl Zeiss AG, Oberkochen, Germany) equipped with
a Micromax 512.times.768 CCD camera (Princeton instruments,
Trenton, N.J.) through a XF43 filter set (PB546/FT580/LP590, Omega
Optical, Brattleboro, Vt.) with a 5 second exposure.
FIGS. 4 and 5 show the hybridisation results for colony generation
on tubes functionalised with either; FIG. 4 oligonucleotide p57 or
FIG. 5 oligonucleotide p58. The control reaction shows very few
fluorescent spots, since the sequence of the flanking regions on
the template do not correspond to the primer sequences grafted onto
the well. In contrast, FIG. 5 shows the number of fluorescent spots
detected when the primers grafted to the wells match the flanking
sequences on the initiating DNA template. Calculating the number of
fluorescent spots detected and taking into consideration the
magnification, we can estimate that there are between 3 and
5.times.10.sup.7 colonies/cm.sup.2. The photos are generated by the
program, Winview 1.6.2 (Princeton Instruments, Trenton, N.J.) with
backgrounds and intensities normalised to the same values.
B) SCHEME SHOWING THE SIMULTANEOUS AMPLIFICATION AND IMMOBILISATION
OF NUCLEIC ACID MOLECULES USING TWO DIFFERENT TYPES OF PRIMER
Referring now to FIGS. 6A and 6B, another embodiment of the present
invention is illustrated. Here two different immobilised primers
are used to provide primer extension.
In this embodiment the target molecule shown is provided with a
nucleotide sequence at its 3' end (AAT-3') that is complementary to
the sequence of a first primer, (5'-ATT, I), which is grafted on
the surface, so that annealing with that primer can occur. The
sequence (5'-GGT) at the 5' end of the target molecule, III,
corresponds to the sequence (5'-GGT) of a second primer, II, which
is also grafted to the surface, so that the sequence which is
complementary to the sequence at the 5' end can anneal with that
said second primer. Generally said complementary sequence (5'-ACC)
is chosen so that it will not anneal with the first primer
(5'-ATT). Unlike the situation described in section A, once the 3'
end of a newly synthesised strand anneals to a primer on the
surface, it will have to find a primer whose sequence is different
from the sequence it carries at its 5' end (see the difference
between FIG. 1A, (f) and FIG. 6A, (f)).
The embodiment shown in FIGS. 6A and 6B have an advantage over the
embodiment illustrated in FIG. 1 since the possibility of one end
of a single stranded target molecule annealing with another end of
the same molecule in solution can be avoided and therefore
amplification can proceed further. The possibility of annealing
occurring between both ends of an immobilised complement to a
target molecule can also be avoided.
Example 2
A mix of two oligonucleotides which are phosphorylated at the
5'-end (Microsynth GmbH, Balgach, Switzerland) have been grafted on
96 well Nucleolink plates (Nunc, Denmark) as recommended by the
manufacturer. The resulting plates has been stored dry at 4.degree.
C. The sequence of the primer, P1, was 5'-GCGCGTAATACGACTCACTA (SEQ
ID NO:5), the sequence of the other primer, P2, was
5'-CGCAATTAACCCTCACTAAA (SEQ ID NO:6). These plates are specially
formulated by Nunc, allowing the covalent grafting of
5'-phosphorylated DNA fragments through a standard procedure.
A template has been cloned in a vector (pBlueScript Skminus,
Stratagene Inc, San Diego, Calif.) with the appropriate DNA
sequence at the cloning site (i.e., corresponding to P1 and P2 at
position 621 and 794 respectively), and 174 bp long linear double
stranded DNA template has been obtained by PCR amplification, using
P1 and P2. The template PCR product has been purified on Qiagen
Qia-quick columns (Qiagen GmbH, Hilden, Germany) in order to remove
the nucleotides and the primers used during the PCR
amplification.
The purified template (in 50 .mu.l solution containing 1.times.PCR
buffer (Perkin Elmer, Foster City, Calif.) with the four
deoxyribonucleoside triphosphates (dNTPs) at 0.2 mM, (Pharmacia,
Uppsala, Sweden) and 2.5 units of AmpliTaq Gold DNA polymerase
(Perkin Elmer, Foster City, Calif.)) has been spread on the
support, i.e. on the Nucleolink plates grafted with P1 and P2 (the
plates have been rinsed with a solution containing 100 mM TRIS-HCl
(pH 7.5), 150 mM NaCl and 0.1% Tween 20 (Fluka, Switzerland) at
room temperature for 15 min). This solution has been incubated at
93.degree. C. for 9 minutes to activate the DNA polymerase and then
60 cycles (94.degree. C./30 sec., 48.degree. C./30 sec., 72.degree.
C./30 sec.) have been performed on a PTC 200 thermocycler. Several
different concentrations of PCR template have been tested
(approximately 1, 0.5, 0.25, 0.125, 0.0625 ng/.mu.l) and for each
sample a control reaction carried out without Taq polymerase has
been performed (same conditions as above but without DNA
polymerase).
Each sample has been stained with YO-PRO (Molecular Probes,
Portland Oreg.), a highly sensitive stain for double stranded DNA.
The resulting products have been observed on a confocal microscope
using a 40.times. objective (LSM 410, Carl Zeiss AG, Oberkochen,
Germany) with appropriate excitation (an 488 argon laser) and
detection filters (510 low pass filter) (note: the bottom of each
well is flat and allows observation with an inverted fluorescence
microscope).
In FIG. 7, the control well (without added DNA template, panel a)
shows only rare objects which can be observed on a blank surface
(these objects were useful at this stage for reporting that the
focus was correct). These objects have an irregular shape, are 20
to 100 micrometers in size and have a thickness much larger than
the field depth of the observation. In a well where DNA polymerase
was present (FIG. 7, panel ii), in addition to the objects of
irregular shape observed in the control well, a great number of
fluorescent spots can be observed. They present a circular shape,
they are 1 to 5 micro meters in size and do not span the field of
view. The number of spots depends on the concentration of the
template used for initiating colony formation. From the observed
size of the colonies, one can estimate that more than 10,000
distinct colonies can be arrayed within 1 mm.sup.2 of support.
Example 3
Oligonucleotides (Microsynth GmbH, Switzerland) were grafted onto
Nucleolink wells (Nunc, Denmark). Oligonucleotide P1 corresponds to
the sequence 5'-TTTTTTCTCACTATAGGGCGAATTGG (SEQ ID NO:7) and
oligonucleotide P2 corresponds to 5'-TTTTTTCTCACTAAAGGGAACAAAAGCTGG
(SEQ ID NO:8). In each Nucleolink well, a 45 .mu.l of 10 mM
1-methyl-imidazole (pH 7.0) (Sigma Chemicals, St. Louis, Mo.)
solution containing 360 fmol of P1 and 360 fmol of P2 was added. To
each well, 15 .mu.l of 40 mM
1-ethyl-3-(3dimethylaminopropyl)-carbodiimide (pH 7.0) (Sigma
Chemicals) in 10 mM 1-methyl-imidazole, was added to the solution
of oligonucleotides. The wells were then sealed and incubated at
50.degree. C. for 16 hours. After the incubation, wells have been
rinsed twice with 200 .mu.l of RS (0.4N NaOH, 0.25% Tween 20),
incubated 15 minutes with 200 .mu.l RS, washed twice with 200 .mu.l
RS, and twice with 200 .mu.l TNT (100 mM Tris/HCl pH7.5, 150 mM
NaCl, 0.1% Tween 20), before they are put to dry at 50.degree. C.
in an oven. The dried tubes were stored in a sealed plastic bag at
4.degree. C.
Colony growing was initiated in each well with 15 .mu.l of
initiation mix (1.times.PCR buffer, 0.2 mM dNTPs and 0.75 units of
AmpliTaq Gold DNA polymerase, 20 nanograms of template DNA, where
the template DNA was either S1 DNA or S2 DNA or a mixture of
different ratios of S1 DNA and S2 DNA, as indicated in discussion
to FIG. 6B. S1 and S2 are 704 base pair and 658 bp fragments,
respectively, which have been cloned into pBlueScript Skminus
plasmids and subsequently amplified through a PCR using P1 and P2
as primers. The fragments were purified on Qiagen Qia-quick columns
(QIAGEN GmbH, Germany) in order to remove the nucleotides and the
primers.
Each well was sealed with Cycleseal.TM. (Robbins Scientific Corp.,
Sunnyvale, Calif.), and incubated at 93.degree. C. for 9 minutes,
65.degree. C. for 5 minutes and 72.degree. C. for 2 minutes and
back to 93.degree. C. Then 200 .mu.l TNT solution was used in three
successive one minute long washes at 93.degree. C. The initiation
mix was then replaced by 15 .mu.l growing mix (same as initiation
mix, but without template DNA), and growing was performed by
incubating the sealed wells 9 minutes at 93.degree. C. and
repeating 40 times the following conditions: 93.degree. C. for 45
seconds, 65.degree. C. for 3 minutes, 72.degree. C. for 2 minutes.
After completion of this program, the wells were kept at 6.degree.
C. until further use. The temperature control was performed in a
PTC 200 thermo-cycler, using the silicon pad provided in the
Nucleolink kit and the heated (1040 C) lid of the PTC 200.
A 640 base pair fragment corresponding to the central sequence of
the S1 fragment, but not including the P1 or P2 sequence was
amplified by PCR as previously described. The probe was labeled
with biotin-16-dUTP (Boehringer-Mannheim, Germany) using the
Prime-it II random primer labeling kit (Stratagene, San Diego,
Calif.) according to the manufacturers instructions.
The biotinylated probes were hybridized to the samples in EasyHyb
buffer (Boehringer-Mannheim, Germany), using the following
temperature scheme (in the PTC 200 thermocycler): 94.degree. C. for
5 minutes, followed by 68 steps of 0.5.degree. C. decrease in
temperature every 30 seconds (in other words, the temperature is
decreased down to 60.degree. C. in 34 minutes), using sealed wells.
The samples are then washed 3 times with 200 .mu.l of TNT at room
temperature. The wells are then incubated for 30 minutes with 50
.mu.l TNT containing 0.1 mg/ml BSA. Then the wells are incubated 5
minutes with 15 .mu.l of solution of red fluorescent,
Neutravidin-coated, 40 nm FluoSpheres.RTM. (580 nm excitation and
605 nm emission, Molecular Probes, Portland, Oreg.). The solution
of microspheres is made of 2 .mu.l of the stock solution of
microspheres, which have been sonicated for 5 minutes in a 50 W
ultra-sound water-bath (Elgasonic, Bienne, Switzerland), diluted in
1 ml of TNT solution containing 0.1 mg/ml BSA and filtered with
Millex GV4 0.22 .mu.m pore size filter (Millipore, Bedford,
Mass.).
The stained samples are observed using an inverted Axiovert 10
microscope using a 20.times. objective (Carl Zeiss AG, Oberkochen,
Germany) equipped with a Micromax 512.times.768 CCD camera
(Princeton Instruments, Trenton, N.J.), using a XF43 filter set
(PB546/FT580/LP590, Omega Optical, Brattleboro, Vt.), and 10
seconds of light collection. The files are converted to TIFF format
and processed in the suitable software (PhotoPaint, Corel Corp.,
Ottawa, Canada). The processing consisted in inversion and linear
contrast enhancement, in order to provide a picture suitable for
black and white print-out on a laser printer.
The FIG. 8 shows the results for 3 different ratios of the S1/S2
templates used in the initiating reaction: i) the S1/S2 is 1/0,
many spots can be observed, ii) the S1/S2 is 1/10, and the number
of spots is approximately 1/10 of the number of spots which can be
observed in the i) image, as expected, and iii) the S1/S2 is 0/1,
and only a few rare spots can be seen.
C) SCHEME SHOWING SIMULTANEOUS AMPLIFICATION AND IMMOBILISATION OF
NUCLEIC ACID MOLECULES WHEN THE TARGET MOLECULE CONTAINS INTERNAL
SEQUENCES COMPLEMENTARY TO THE IMMOBILISED PRIMERS
FIGS. 9A and 9B are provided to show that the sequences shown at
the 5' and 3' ends of the target molecule illustrated in FIGS. 1A
and 1B and FIGS. 6A and 6B need not be located at the ends of a
target molecule.
A target nucleic acid molecule (II) may have a sequence at each (or
either) end that is neither involved in annealing with a primer nor
in acting as a template to provide a complementary sequence that
anneals with a primer (sequence 5'-AAA and sequence 5'-CCC) One of
the internal sequences (5'-AAT) is used as a template to synthesise
a complementary sequence, III, thereto (5'-TTT), as is clear from
FIGS. 9A, (a) to (e).
The sequence 5'-TTT is not however itself used to provide a
sequence complementary thereto, as is clear from FIGS. 9A, (f) to
(h) and FIG. 9B, (i) to (k). It can be seen from FIG. 9B, (1) that
only one of the four immobilised strands shown after two rounds of
primer extension and a strand separation step comprises the
additional sequence 5'-TTT and that no strand comprising a
complementary sequence (5'-AAA) to this sequence is present (i.e.
only one strand significantly larger than the others is present).
After several rounds of amplification the strand comprising the
sequence 5'-TTT will represent an insignificant proportion of the
total number of extended, immobilised nucleic acid molecules
present.
D) USING NUCLEIC ACID STRANDS PRESENT IN COLONIES TO SYNTHESISE
ADDITIONAL COPIES OF NUCLEIC ACID STRANDS
Amplified, single stranded nucleic acid molecules present in
colonies provided by the present invention can themselves be used
as templates to synthesise additional nucleic acid strands.
FIGS. 10A and 10B illustrate one method of synthesising additional
nucleic acids using immobilised nucleic acids as a starting
point.
Colonies will usually comprise both a given nucleic acid strand and
its complement in immobilised form (FIG. 10A, (a)). Thus they can
be used to provide additional copies not only of a given nucleic
acid strand but also of its complement.
One way of doing this is to provide one or more primers (primers
TTA and TGG) in solution that anneal to amplified, immobilised
nucleic acid strands present in colonies (FIG. 10A (c)) provided by
the present invention. (These primers may be the same as primers
initially used to provide the immobilised colonies, apart from
being provided in free rather than immobilised form.) The original
DNA colony is denatured by heat to it single-stranded form (FIG.
10A (b)) allowing primers TTA and TGG to anneal to the available 3'
end of each DNA strand. Primer extension, using AmpliTaq DNA
polymerase and the four deoxyribonucleoside triphosphates (labeled
or unlabeled) can then be used to synthesise complementary strands
to immobilised nucleic acid strands or at least to parts thereof
(step (iii)).
Once newly formed strands (FIG. 10B (d)) have been synthesised by
the process described above, they can be separated from the
immobilised strands to which they are hybridised (e.g. by heating).
The process can then be repeated if desired using the PCR reaction,
to provide large number of such strands in solution (FIG. 10B,
(e).
Strands synthesised in this manner, after separation from the
immobilised strands, can, if desired, be annealed to one another
(i.e. a given strand and its complement can anneal) to provide
double-stranded nucleic acid molecules in solution. Alternatively
they can be separated from one another to provide homogenous
populations of single-stranded nucleic acid molecules in
solution.
It should also be noted that once single-stranded molecules are
provided in solution they can be used as templates for PCR (or
reverse PCR). Therefore it is not essential to continue to use the
immobilised nucleic acid strands to obtain further amplification of
given strands or complementary strands thereto.
It should be noted that where a plurality of colonies are provided
and nucleic acid strands in different colonies have different
sequences, it is possible to select only certain colonies for use
as templates in the synthesis of additional nucleic acid molecules.
This can be done by using primers for primer extension that are
specific for molecules present in selected colonies.
Alternatively primers can be provided to allow several or all of
the colonies to be used as templates. Such primers may be a mixture
of many different primers (e.g. a mixture of all of the primers
originally used to provide all of the colonies, but with the
primers being provided in solution rather than in immobilised
form).
Example 4
Oligonucleotides (Microsynth GmbH Balgach, Switzerland) were
grafted onto Nucleolink wells (Nunc, Denmark). Oligonucleotide P1
corresponds to the sequence 5'-TTTTTTTTTTCACCAACCCARACCAACCCAAACC
(SEQ ID NO:9) and oligonucleotide P2 corresponds to
5TTTTTTTTTTAGAAGGAGAAGGAAAGGGAAAGGG (SEQ ID NO:10). In each
Nucleolink well, a 45 .mu.l of 10 mM 1-methyl-imidazole (pH 7.0)
(Sigma Chemicals) solution containing 360 fmol of P1 and 360 fmol
of P2 was added. To each well, 15 .mu.l of 40 mM
1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (pH 7.0) (Sigma
Chemicals) in 10 mM 1-methyl-imidazole, was added to the solution
of oligonucleotides. The wells were then sealed and incubated at
50.degree. C. for 16 hours. After the incubation, wells have been
rinsed twice with 200 .mu.l of RS (0.4N NaOH, 0.25% Tween 20),
incubated 15 minutes with 200 .mu.l RS, washed twice with 200 .mu.l
RS, and twice with 200 .mu.l TNT (100 mM Tris/HCl pH7.5, 150 mM
NaCl, 0.1% Tween 20), before they are put to dry at 50.degree. C.
in an oven. The dried tubes were stored in a sealed plastic bag at
4.degree. C.
Colony growing was initiated in each well with 15 .mu.l of
initiation mix (1.times.PCR buffer, 0.2 mM dNTPs and 0.75 units of
AmpliTaq DNA polymerase, 20 nanograms of template DNA, where the
template DNA was either S1 DNA or S2 DNA or a 1/1 mixture of S1 DNA
and S2 DNA, as indicated in discussion to Example 3. S1 and S2 are
658 base pair and 704 b.p. fragments, respectively, which have been
prepared as described in EXAMPLE 3.
Each well was sealed with Cycleseal.TM. (Robbins Scientific Corp.,
Sunnyvale, Calif.) and incubated at 93.degree. C. for 9 minutes,
65.degree. C. for 5 minutes and 72.degree. C. for 2 minutes and
back to 93.degree. C. Then 200 .mu.l TNT solution was used in three
successive one minute long washes at 93.degree. C. The initiation
mix was then replaced by 15 .mu.l growing mix (same as initiation
mix, but without template DNA) and growing was performed by
incubating the sealed wells 9 minutes at 93.degree. C. and
repeating 40 times the following conditions: 93.degree. C. for 45
seconds, 65.degree. C. for 3 minutes, 72.degree. C. for 2 minutes.
After completion of this program, the wells were kept at 6.degree.
C. until further use. The temperature control was performed in a
PTC 200 thermo-cycler.
Different treatments where applied to 6 sets (A, B, C, D, E and F)
at 6.degree. C. of 3 wells (1, 2, 3), one prepared with template
S1, one with template S1 and template S2 and one prepared with
template S2 alone (yielding A1, A2, A3, . . . , F1, F2, F3). The
set A was left untreated, set B has been incubated for 10 minutes
with BAL-31 exonuclease (New England Biolabs, Beverly, Mass.) at
37.degree. C. in BAL-31 buffer (BAL-31 essentially digests double
stranded DNA which has both ends free), set C has been incubated
for 10 minutes with S1 nuclease (Pharmacia, Uppsala, Sweden) at
37.degree. C. in S1 buffer (S1 nuclease essentially digests single
stranded DNA), set D, E and F have been incubated with both BAL-31
and S1 nucleases. Reactions were stopped by rinsing the wells with
TNT buffer.
PCR (25 cycles, 30 sec. at 94.degree. C., 45.degree. sec. at
60.degree. C., 45 sec. at 72.degree. C.) has been performed in the
Nucleolink wells with 0.25 pM primers P70
(5'-CACCAACCCAAACCAACCCAAACCACGACTCACTATAGGGCGAA) (SEQ ID NO:11)
and P71 (5' AGAAGGAGAAGGAAAGGGAAAGGGTAAAGGGAACAAAAGCTGGA) (SEQ ID
NO:12) in solution in sets A, B, C and D. P70 and P71 are suited
for the amplification of both S1 and S2, since primer P70 contains
the sequence of primer P1 and p71 contains P2. In the set E wells,
PCR has been performed with a set of forward (P150,
5'-GGTGCTGGTCCTCAGTCTGT) (SEQ ID NO:13) and reverse (P151, 5'
CCCGCTTACCAGTTTCCATT) (SEQ ID NO:14) primers which are within S1
and not within S2 so as to produce a 321 bp PCR product, and in the
set F wells, PCR has been performed with a set of forward (P152, 5'
CTGGCCTTATCCCTAACAGC) (SEQ ID NO:15) and reverse (P153,
5'-CGATCTTGGCTCATCACAAT) (SEQ ID NO:16) primers which are within S2
and not within S1 so as to produce a 390 bp PCR product. For each
of the 18 PCR reactions, 3 .mu.l of solution have been used for gel
electrophoresis on 1% agarose in presence of 0.1 .mu.g/ml
ethidium-bromide. The pictures of the gels are presented in FIGS.
11A and 11B. These pictures show that DNA in the colonies is
protected from exonuclease digestion (sets B, C and D as compared
to set A), and that both S1 and S2 can be recovered either
simultaneously using P1 and P2 (sets A, B, C and D) or specifically
(set E and F). In set E and F, where the shorter PCR products are
more efficiently amplified than the longer PCR products in sets A,
B, C, D, a cross-contamination between the S1 and S2 templates is
detectable (see lane E2 and F1).
E) PROVISION OF SECONDARY COLONIES
It is also possible to modify initially formed colonies to provide
different colonies (i.e. to provide colonies comprising immobilised
nucleic acid molecules with different sequences from those
molecules present in the initially formed colonies). Here, the
initially formed colonies are referred to as "primary colonies" and
the later formed colonies as "secondary colonies". A preliminary
procedure is necessary to turn the primary colonies into "secondary
primers" which will be suitable for secondary colony
generation.
FIGS. 12A, 12B, and 12C shows how `secondary primers` are generated
using existing primary colonies. As a starting point, the primary
colony (FIG. 12A, (a)) is left in the fully hybridised,
double-stranded form. A single-strand specific DNA exonuclease
might be used to remove all primers which have not been elongated.
One could also choose to cap all free 3'-OH ends of primers with
dideoxyribonucleotide triphosphates using a DNA terminal
transferase (step (i), FIG. 12A, (b)).
Secondly and independently, the DNA molecules forming the colonies
can be cleaved by using endonucleases. For example, a restriction
enzyme that recognises a specific site within the colony (depicted
by the `RE` arrow in FIG. 12B (c)) and cleaves the DNA colony (step
(ii), FIG. 12B). If desired, the enzymatically cleaved colony (FIG.
12B, (d)) can then be partially digested with a 3' to 5'
double-strand specific exonuclease (e.g. E. coli exonuclease III,
depicted by `N`, step (iii), FIG. 12B, (d)). In any case, the
secondary primers are available after denaturation (e.g., by heat)
and washing (FIG. 12B, (e)).
Alternatively, the double stranded DNA forming the colonies (FIG.
12C, (f)) can be digested with the double-strand specific 3'-5'
exonuclease, which digest only one strand of double stranded DNA.
An important case is when the exonuclease digests only a few bases
of the DNA molecule before being released in solution, and when
digestion can proceed when another enzyme binds to the DNA molecule
(FIG. 12C, (g)). In this case the exonuclease digestion will
proceed until there remain only single stranded molecules which, on
average, are half the length of the starting material, and are
without any complementary parts (which could form partial duplexes)
remaining in the single stranded molecules in a colony (FIG.
12C(h)).
In all cases, these treatments result in single-stranded fragments
grafted onto a support which correspond to the sequence of the
original template and that can be used for new DNA colony growing
if an appropriate new template is provided for colony initiation
(FIG. 12B, (e) and FIG. 12C, (h)).
The result of such a treatment, thus a support holding secondary
primers, will be referred to as a "support for secondary colony
growing". Templates useful for secondary colony growing may include
molecules having known sequences (or complements of such
sequences). Alternatively templates may be derived from unsequenced
molecules (e.g. random fragments). In either event the templates
should be provided with one or more regions for annealing with
nucleic acid strands present in the primary colonies.
FIGS. 13A and 13B show how a secondary colony can be generated when
an appropriate template (TP, FIG. 13A, (a)) is provided for a
second round of DNA colony generation on a support for secondary
colony growing, holding secondary primers. In this example,
treatment of the primary colony as described above has generated
the secondary primers, SP1 and SP2 (FIG. 13A, (a)). The template
TP, will hybridise to its complementary secondary primer, SP1, and
following an extension reaction using a DNA polymerase as
described, will be extended as depicted (FIG. 13A, (b)). Following
a denaturing (step ii), reannealing (step iii) and DNA polymerase
(step iv) cycle, a replica of the original primary colony will be
formed (FIG. 13B, (e)).
The maximum size of a secondary colony provided by this embodiment
of the present invention is restricted by the size of the primary
colony onto which it grows. Several secondary growing processes can
be used sequentially to create colonies for specific applications
(i.e. a first colony can be replaced with a second colony, the
second colony can be replaced with a third colony, etc.)
F) PROVISION OF EXTENDED PRIMERS
FIGS. 14A and 14B show how extended primers can be generated on an
array of oligonucleotides. The same procedure could be applied to a
support covered with colonies or secondary primers as described in
section E.
In FIG. 14A, (a) a support is provided having a plurality of
immobilised primers shown thereon. Different immobilised primers
are shown present in different regions of the support (represented
by squares). Primers having the sequence 5'-AAA are present in one
square and primers having the sequence 5'-GGG are present in
another square.
FIG. 14A, (b) and FIG. 14B, (c) and (d) show how the initial
primers present (initial primers) are modified to give different
primers (extended primers). In this example, those initial primers
having the sequence 5'-AAA are modified to produce two different
types of extended primers, having the sequences 5'-AAAGCC and
5'-AAATAC respectively. This is achieved by the hybridisation of
oligonucleotide templates, 5'-GTATTT and 5' GGCTTT to the primary
primers immobilised on the surface (FIG. 14A, (b)), followed by DNA
polymerase reaction. Those initial primers having the sequence
5'-GGG are modified to produce two different types of extended
primers, having the sequences 5'-GGGTAT and 5' GGGTAA (FIG. 14A,
(d)) in a similar manner.
The technique of producing extended primers is useful for
transforming immobilised oligonucleotides provided on a DNA chip or
other surface into immobilised primers useful in amplifying a
particular target nucleic acid sequence and/or in amplifying a
complementary strand thereto.
G) PREPARATION OF NUCLEIC ACID FRAGMENTS
Apparatuses of the present invention can be used for various
procedures some of which will be described later on. Nucleic acid
fragments for use in colony generation may be prepared differently
for the different procedures (referred to herein as "prepared
nucleic acids"). Various preparation procedures are described
below:
(i) Preparation of Random DNA Fragments
Here is described a method to prepare DNA originating from one
biological sample (or from a plurality of samples) for
amplification in the case where it is not necessary to keep track
of the origin of the DNA when it is incorporated within a
colony.
The DNA of interest is first extracted from the biological sample
and cut randomly into "small" pieces (e.g., 50 to 10,000 bases
long, but preferentially 500 to 1000 base pairs in length,
represented by bar `I`, FIG. 15, (a)). (This can be done e.g., by a
phenol-chloroform extraction followed by ultrasound treatment,
mechanical shearing, by partial digestion with frequent cutter
restriction endonucleases or other methods known by those skilled
in the art). In order to standardise experimental conditions, the
extracted and cut DNA fragments can be size-fractionated, e.g., by
agarose gel electrophoresis, sucrose gradient centrifugation or gel
chromatography. Fragments obtained within a single fraction can be
used in providing templates in order to reduce the variability in
size of the templates.
Secondly, the extracted, cut and (optionally) sorted template DNA
fragments can be ligated with oligonucleotide linkers (IIa and IIb,
FIG. 15, (a)) containing the sequence of the primer(s) which have
previously been grafted onto a support. This can be achieved, for
instance, using "blunt-end" ligation. Alternatively, the template
DNA fragments can be inserted into a biological vector at a site
that is flanked by the sequence of the primers that are grafted on
the support. This cloned DNA can be amplified within a biological
host and extracted. Obviously, if one is working with a single
primer grafted to the solid support for DNA colony formation,
purifying fragments containing both P1 and P2 primers does not pose
a problem. Hereafter, the DNA fragments obtained after such a
suitable process are designated by the expression: "prepared
genomic DNA" (III, FIG. 15, (a)).
(ii) Preparation of Random DNA Fragments Originating from a
Plurality of Samples
Here it is described how to prepare DNA originating from a
plurality of biological samples in the case where it is necessary
to keep track of the origin of the DNA when it is incorporated
within a colony.
The procedure is the same as that described in the previous section
except that in this case, the oligonucleotide linkers used to tail
the randomly cut genomic DNA fragments are now made of two parts;
the sequence of the primers grafted onto the surface (P1 and P2,
FIG. 15 (b)) and a "tag" sequence which is different for each
sample and which will be used for identifying the origin of the DNA
colony. Note that for each sample, the tag may not be unique, but a
plurality of tags could be used. Hereafter, we will designate the
DNA fragments obtained after such a suitable process by the
expression "tagged genomic DNA" (III, FIG. 15, (b)).
This tagging procedure can be used for providing colonies carrying
a means of identification which is independent from the sequence
carried by the template itself. This could also be useful when some
colonies are to be recovered specifically (using the procedure
given in section D). This could also be useful in the case the
recovered colonies are further processed, e.g., by creating new
primary colonies and a cross reference between the original
colonies and the new colonies is desired.
(iii) Preparation of DNA Fragments Corresponding to a Plurality of
DNA Sequences Originating from One Sample
The DNA of interest can first be extracted from a biological sample
by any means known by those skilled in the art (as mentioned
supra). Then the specific sequences of interest can be amplified
with PCR (step (i), FIG. 15 (c)) using PCR primers (IIa and IIb)
made of two parts; 1) at the 5'-end, the sequences corresponding to
the sequences of primer oligonucleotide(s) that have been grafted
onto a surface (P1 and P2) and 2) at the 3'-end, primer sequences
specific to the sequence of interest (S1 and S2). Hereafter, we
will designate the DNA fragments obtained after such a suitable
process by the expression: "prepared DNA" (III, FIG. 15 (c)).
(iv) Preparation of a Plurality of DNA Fragments Originating from a
Plurality of Samples
The procedure is the same as in the previous section except that in
this case the DNA primers (IIa and IIb) used to perform the PCR
amplification (step (i), FIG. 13, (D)) are now made of three parts;
1) the sequence of the primers grafted onto the surface (P1 and
P2), 2) a "tag" sequence which is different for each sample and
which will be used for the identifying the origin of the DNA colony
and 3) primer sequences surrounding the specific sequence of
interest (S1 and S2). Note that for each sample, a plurality of
tags might be used, as in (ii) supra.
Hereafter, we will designate the DNA fragments obtained after such
a suitable process by the expression: "tagged DNA" (III, FIG. 13,
(d)). Potential uses of tags are the same as in (ii), supra.
(v) Preparation of mRNA
The procedure is similar to the procedures described for preparing
DNA fragments in the previous sections except that the starting
point is to extract mRNA by any means known to those skilled in the
art (e.g., by use of commercially available mRNA preparation kits).
The mRNA can be copied into double-stranded cDNA by any means known
to those skilled in the art (e.g. by using a reverse transcriptase
and a DNA polymerase). Certainly, the tags and primers described
supra can be used in conjunction with the process of
double-stranded cDNA synthesis to allow their incorporation into
the templates. Hereafter, we will designate the mRNA fragments
obtained after such suitable processes by the expressions:
"prepared total mRNA" (cf. "prepared genomic DNA", as described in
section (I) supra), "tagged total mRNA", (cf. "tagged genomic DNA",
as described in section (ii) supra), "prepared mRNA" (cf. "prepared
DNA", as described in section (iii) supra) and "tagged mRNA" (cf.
"tagged DNA", as described in section (iv) supra).
H) PREFERRED DETECTION ASSAYS
In assay procedures of the present invention labels may be used to
provide detectable signals. Examples include: a) a fluorescent
group or an energy-transfer based fluorescence system. b) a biotin
based system. In this case colonies can be incubated with
streptavidin labeled with a fluorescent group or an enzyme (e.g.
fluorescent latex beads coated with streptavidin; streptavidin
labeled with fluorescent groups; enzymes for use with the
corresponding fluorescence assay). c) a system based on detecting
an antigen or a fragment thereof--e.g. a hapten (including biotin
and fluorescent groups). In this case colonies can be incubated
with antibodies (e.g. specific for a hapten). The antibodies can be
labeled with a fluorescent group or with an enzyme (e.g.
fluorescent latex beads coated with the antibody; antibodies
labeled with fluorescent groups; antibodies linked to an enzyme for
use with a corresponding fluorescence or luminescence assay, etc.).
d) a radio-label (e.g. incorporated by using a 5'-polynucleotide
kinase and [.gamma.-.sup.32P] adenosine triphosphate or a DNA
polymerase and [.alpha.-.sup.32P or .alpha.-.sup.33P]
deoxyribonucleoside triphosphates to add a radioactive phosphate
group(s) to a nucleic acid). Here colonies can be incubated with
scintillation liquid. e) a dye or other staining agent.
Labels for use in the present invention are preferably attached a)
to nucleic acids b) to proteins which bind specifically to double
stranded DNA (e.g., histones, repressors, enhancers) and/or c) to
proteins which bind specifically to single stranded DNA (e.g.
single-stranded nucleic acid binding protein).
Labeled colonies are preferably detected by: a) measuring
fluorescence. b) measuring luminescence. c) measuring radioactivity
d) measuring flow or electric field induced fluorescence
anisotropy. and/or e) measuring the polymer layer thickness.
Staining agents can be used in the present invention. Thus DNA
colonies can be incubated with a suitable DNA-specific staining
agent, such as the intercalating dyes, ethidium bromide, YO-YO,
YO-PRO (Molecular Probes, Eugene, Oreg.).
With certain staining agents the result can be observed with a
suitable fluorescence imaging apparatus.
Examples of particular assays/procedures will now be described in
greater detail:
I) PREFERRED EMBODIMENTS OF ASSAYS OF THE PRESENT INVENTION
(i) Nucleic Acid Probe Hybridisation Assay
DNA colonies are first prepared for hybridisation. Then they are
hybridised with a probe (labeled or unlabelled). If required, the
hybridised probed is assayed, and the result is observed. This can
be done with an apparatus of the present invention (e.g. as
described supra)
Preparation for Hybridization
In a preferred embodiment of the present invention colonies are
treated with a DNA restriction endonuclease which is specific
either for a sequence provided by a double stranded form of one of
the primers originally grafted onto the surface where colonies are
formed or for another sequence present in a template DNA molecule
(see e.g. FIG. 12B, (c)).
After restriction enzyme digestion, the colonies can be heated to a
temperature high enough for double stranded DNA molecules to be
separated. After this thermal denaturing step, the colonies can be
washed to remove the non-hybridised, detached single-stranded DNA
strands, leaving a remaining attached single-strand DNA.
In another embodiment the colonies can be partially digested with a
double-strand specific 3' to 5' DNA exonuclease (see section E,
FIG. 12C, (f)) which removes one strand of DNA duplexes starting
from the 3' end, thus leaving a part of a DNA molecule in single
stranded form.
Alternatively, DNA in colonies can first be heat denatured and then
partially digested with an single-strand specific 3' to 5' DNA
exonuclease which digests single stranded DNA starting from the 3'
end.
A further alternative is simply to heat denature DNA in the
colonies.
Hybridisation of the Probe
Single-stranded nucleic acid probes (labeled or unlabelled) can be
hybridised to single-stranded DNA in colonies at the appropriate
temperature and buffer conditions (which depends on the sequence of
each probe, and can be determined using protocols known to those
skilled in the art).
Assaying of Unlabelled Hybridised Probes
A hybridised probe provided initially in unlabelled form can be
used as a primer for the incorporation of the different (or a
subset of the different) labeled (or a mix of labeled and
unlabelled) deoxyribonucleoside triphosphates with a DNA
polymerase. The incorporated labeled nucleotides can then be
detected as described supra.
Cyclic Assaying of Labeled or Unlabelled Probes
Firstly, the DNA colonies can be prepared for hybridisation by the
methods described supra. Then they can be hybridised with a probe
(labeled or initially unlabelled). If required, hybridised labeled
probes are assayed and the result is observed with an apparatus as
described previously. The probe may then be removed by heat
denaturing and a probe specific for a second DNA sequence may be
hybridised and detected. These steps maybe repeated with new probes
as many times as desired.
Secondly, the probes can be assayed as described supra for
unlabelled probes, except that only a subset (preferably 1 only) of
the different (labeled or unlabelled) nucleotides are used at each
cycle. The colonies can then be assayed for monitoring the
incorporation of the nucleotides. This second process can be
repeated until a sequence of a desired length has been
determined.
(ii) In Situ RNA Synthesis Assay
In this embodiment, DNA colonies can be used as templates for in
situ RNA synthesis as depicted in FIG. 16, (a). DNA colonies can be
generated from templates and primers, such that a RNA polymerase
promoter sequence is positioned at one end of the double-stranded
DNA in the colony. DNA colonies can then be incubated with RNA
polymerase and the newly synthesised RNA (cRNA) can be assayed as
desired. The detection can be done non-specifically (e.g.,
staining) or in a sequence dependent way (e.g., hybridisation).
The DNA template (I, FIG. 16, (a)) to be amplified into a colony is
generated by PCR reaction using primers (IIa and IIb) which have
the following four parts; 1) sequence identical to the sequences of
the primers grafted onto the surface (`P1` and P2'), 2) a "tag"
sequence which is different for each sample, a sequence
corresponding to a RNA polymerase promoter, i.e., the T3, T7 and
SP6 RNA promoters, (`RPP`, FIG. 16, (a)) and 4) primer sequences
surrounding the specific sequence of interest (`S1` and `S2`).
Hereafter, we will designate the DNA fragments obtained after such
a suitable process by the expression: "tagged RNA synthesis DNA"
(III, FIG. 16, (b)).
After amplification of the DNA template from the original DNA
sample, these templates are used to generate DNA colonies. The DNA
colonies (IV, FIG. 16, (c)) are then incubated with the RNA
polymerase specific for the RNA polymerase promoter (`RPP`, FIG. 16
(c)). This will generate a copy of RNA specific for the DNA colony
template (Template-cRNA, V, FIG. 16, (d)).
cRNA thus synthesised can be isolated and used as hybridisation
probes, as messenger RNA (mRNA) templates for in vitro protein
synthesis or as templates for in situ RNA sequence analysis.
(iii) Methods for Sequencing
In another embodiment of the present invention, colonies can be
analysed in order to determine sequences of nucleic acid molecules
which form the colonies. Since very large numbers of the same
nucleic acid molecules can be provided within each colony the
reliability of the sequencing data obtained is likely to be very
high.
The sequences determined may be full or partial. Sequences can be
determined for nucleic acids present in one or more colonies. A
plurality of sequences may be determined at the same time.
In some embodiments the sequence of a complementary strand to a
nucleic acid strand to be sequenced (or of a part thereof) may be
obtained initially. However this sequence can be converted using
base-pairing rules to provide the desired sequence (or a part
thereof). This conversion can be done via a computer or via a
person. It can be done after each step of primer extension or can
be done at a later stage.
Sequencing can be done by various methods. For example methods
relying on sequential restriction endonuclease digestion and linker
ligation can be used. One such method is disclosed in W095/27080
for example. This method comprises the steps of: ligating a probe
to an end of a polynucleotide, the probe having a nuclease
recognition site; identifying one or more nucleotides at the end of
the polynucleotide; and cleaving the polynucleotide with a nuclease
recognising the nuclease recognition site of the probe such that
the polynucleotide is shortened by one or more nucleotides.
However in a preferred method of the present invention, amplified
nucleic acid molecules (preferably in the form of colonies, as
disclosed herein) are sequenced by allowing primers to hybridise
with the nucleic acid molecules, extending the primers and
detecting the nucleotides used in primer extension. Preferably,
after extending a primer by a single nucleotide, the nucleotide is
detected before a further nucleotide is used in primer extension
(step-by-step sequencing).
One or more of the nucleotides used in primer extension may be
labeled. The use of labeled nucleotides during primer extension
facilitates detection. (The term "label" is used in its broad sense
to indicate any moiety that can be identified using an appropriate
detection system. Preferably the label is not present in naturally
occurring nucleotides.) Ideally, labels are non-radioactive, such
as fluorophores. However radioactive labels can be used.
Where nucleotides are provided in labeled form the labels may be
the same for different nucleotides. If the same label is used each
nucleotide incorporation can be used to provide a cumulative
increase of the same signal (e.g. of a signal detected at a
particular wavelength). Alternatively different labels may be used
for each type of nucleotide (which may be detected at different
wavelengths).
Thus four different labels may be provided for dATP, dTTP, dCTP and
dGTP, or the same label may be provided for them all. Similarly,
four different labels may be provided for ATP, UTP, CTP and GTP, or
the same label may be provided for them all). In some embodiments
of the present invention a mixture of labeled and unlabelled
nucleotides may be provided, as will be described in greater detail
later on.
In a preferred embodiment of the present invention the sequencing
of nucleic acid molecules present in at least 2 different colonies
is performed simultaneously. More preferably, sequencing of nucleic
acid molecules present in over 10, over 100, over 1000 or even over
1,000,000 different colonies is performed simultaneously. Thus if
colonies having different nucleic acids molecules are provided,
many different sequences (full or partial) can be determined
simultaneously--i.e. over 10, over 100, over 1000 or even over
1,000,000 different sequences may be determined simultaneously.
If desired, controls may be provided, whereby a plurality of
colonies comprising the same nucleic acid molecules are provided.
By determining whether or not the same sequences are obtained for
nucleic acid molecules in these colonies it can be ascertained
whether or not the sequencing procedure is reliable.
One sequencing method of the present invention is illustrated in
FIG. 17, which is entitled "in situ sequencing". On prepared DNA
colonies hybridised with an appropriate sequencing primer, cyclic
addition of the individual deoxyribonucleoside triphosphates and
DNA polymerase will allow the determination of the DNA sequence
immediately 3' to the sequencing primer. In the example outlined in
FIG. 17, the addition of dGTP allows the determination of colony 1
to contain a `G`. In the second cycle addition of dATP is detected
in both colonies, determining that both colonies have an `A` in the
next position. After several repetitions of the addition of single
deoxyribonucleoside triphosphates, it will be possible to determine
any sequence. For example sequences of at least 10, at least 20, at
least 50 or at least 100 bases may be determined.
If colonies are provided initially in a form comprising
double-stranded molecules the colonies can be processed to provide
single-stranded molecules for use in sequencing as described above.
(It should however be noted that double stranded molecules can be
used for sequencing without such processing. For example a double
stranded DNA molecule can be provided with a promoter sequence and
step-by-step sequencing can then be performed using an RNA
polymerase and labeled ribonucleotides (cf FIG. 16, (d)). Another
alternative is for a nick to be introduced in a double stranded DNA
molecule so that nick translation can be performed using labeled
deoxyribonucleotides and a DNA polymerase with 5' to 3' exonuclease
activity.)
One way of processing double-stranded molecules present in colonies
to provide single-stranded colonies as described later with
reference to FIG. 18. Here double-stranded immobilised molecules
present in a colony (which may be in the form of bridge-like
structures) are cleaved and this is followed by a denaturing step.
(Alternatively a denaturing step could be used initially and could
be followed by a cleavage step). Preferably cleavage is carried out
enzymatically. However other means of cleavage are possible, such
as chemical cleavage. (An appropriate cleavage site can be provided
in said molecule.) Denaturing can be performed by any suitable
means, For example it may be performed by heating and/or by
changing the ionic strength of a medium in the vicinity of the
nucleic acid molecules.
Once single-stranded molecules to be sequenced are provided,
suitable primers for primer extension can be hybridised thereto.
Oligonucleotides are preferred as primers. These are nucleic acid
molecules that are typically 6 to 60, e.g. 15 to 25 nucleotides
long. They may comprise naturally and/or non-naturally occurring
nucleotides. (However other molecules, e.g. longer nucleic acid
strands may alternatively be used as primers, if desired.) The
primers for use in sequencing preferably hybridise to the same
sequences present in amplified nucleic acid molecules as do primers
that were used to provide said amplified nucleic acids. (Primers
having the same/similar sequences can be used for both
amplification and sequencing purposes).
When primers are provided in solution and are annealed (hybridised)
to nucleic acid molecules present in colonies to be sequenced,
those primers which remain in solution or which do not anneal
specifically can be removed after annealing. Preferred annealing
conditions (temperature and buffer composition) prevent
non-specific hybridisation. These may be stringent conditions. Such
conditions would typically be annealing temperatures close to a
primer's Tm (melting temperature) at a given salt concentration
(e.g. 50 nM primer in 200 mM NaCl buffer at 55.degree. C. for a
20-mer oligonucleotide with 50% GC content). (Stringent conditions
for a given system can be determined by a skilled person. They will
depend on the base composition, GC content, the length of the
primer used and the salt concentration. For a 20 base
oligonucleotide of 50% GC, calculated average annealing temperature
is 55-60.degree. C., but in practice may vary between 35 to
70.degree. C.).
Primers used for primer extension need not be provided in solution,
since they can be provided in immobilised form. In this embodiment
the primers should be provided in the vicinity of the immobilised
molecules to which they are to be annealed. (Such primers may
indeed already be present as excess immobilised primers that were
not used in amplifying nucleic acid molecules during the formation
of colonies.)
The nucleic acid molecules present in colonies to be sequenced will
include a sequence that hybridises to the primers to be used in
sequencing (preferably under "stringent" conditions). This portion
can be added to a given molecule prior to amplification (which
molecule may have a totally/partially unknown sequence) using
techniques known to those skilled in the art. For example it can be
synthesised artificially and can be added to a given molecule using
a ligase.
Once a nucleic acid molecule annealed to a primer is provided,
primer extension can be performed. RNA or DNA polymerases can be
used. DNA polymerases are however the enzymes of choice for
preferred embodiments. Several of these are commercially available.
Polymerases which lack 3' to 5' exonuclease activity can be used,
such as T7 DNA polymerase or the small (Klenow) fragment of DNA
polymerase I may be used [e.g. the modified T7 DNA polymerase
Sequenase.TM. 2.0 (Amersham) or Klenow fragment (3' to 5' exo-, New
England Biolabs)]. However it is not essential to use such
polymerases. Indeed, where it is desired that the polymerases have
proof-reading activity polymerases lacking 3' to 5' exonuclease
activity would not be used. Certain applications may require the
use of thermostable polymerases such as ThermoSequenase.TM.
(Amersham) or Taquenase.TM. (ScienTech, St Louis, Mo.). Any
nucleotides may be used for primer extension reactions (whether
naturally occurring or nonnaturally occurring). Preferred
nucleotides are deoxyribonucleotides, dATP, dTTP, dGTP and dCTP
(although for some applications the dTTP analogue dUTP is
preferred) or ribonucleotides ATP, UTP, GTP and CTP; at least some
of which are provided in labeled form.
A washing step is preferably incorporated after each primer
extension step in order to remove unincorporated nucleotides that
may interfere with subsequent steps. The preferred washing solution
should be compatible with polymerase activity and have a salt
concentration that does not interfere with the annealing of primer
molecules to the nucleic acid molecules to be sequenced. (In less
preferred embodiments, the washing solution may interfere with
polymerase activity. Here the washing solution would need to be
removed before further primer extension.)
Considering that many copies of molecules to be sequenced can be
provided in a given colony, a combination of labeled and
non-labeled nucleotides can be used. In this case, even if a small
proportion of the nucleotides are labeled (e.g. fluorescence
labeled), the number of labels incorporated in each colony during
primer extension can be sufficient to be detected by a detection
device. For example the ratio of labeled to non-labeled nucleotides
may be chosen so that, on average, labeled nucleotides are used in
primer extension less than 50%, less than 20%, less than 10% or
even less than 1% of the time (i.e. on average in a given primer
extension step a nucleotide is incorporated in labeled form in less
than 50%, less than 20%, less than 10%, or less than 1% of the
extended primers.)
Thus in a further embodiment of the present invention there is
provided a method for sequencing nucleic acid molecules present in
a colony of the present invention, the method comprising the steps
of: a) providing at least one colony comprising a plurality of
single-stranded nucleic acid molecules that have the same sequences
as one another and that are hybridised to primers in a manner to
allow primer extension in the presence of nucleotides and a-nucleic
acid polymerase; b) providing said at least one colony with a
nucleic acid polymerase and a given nucleotide in labeled and
unlabelled form under conditions that allow extension of the
primers if a complementary base or if a plurality of such bases is
present at the appropriate position in the single stranded nucleic
acid molecules present in said at least one colony; c) detecting
whether or not said labeled nucleotide has been used for primer
extension by determining whether or not the label present on said
nucleotide has been incorporated into extended primers;
Steps b) and c) may be repeated one or more times. Preferably a
plurality of different colonies are provided and several different
sequences are determined simultaneously.
This further embodiment of the present invention can be used to
reduce costs, since relatively few labeled nucleotides are needed.
It can also be used to reduce quenching effects.
It is however also possible to use only labeled nucleotides for
primer extension or to use a major portion thereof (e.g. over 50%,
over 70% or over 90% of the nucleotides used may be labeled). This
can be done for example if labels are selected so as to prevent or
reduce quenching effects. Alternatively labels may be removed or
neutralised at various stages should quenching effects become
problematic (e.g. laser bleaching of fluorophores may be
performed). However this can increase the number of steps required
and it is therefore preferred that labels are not removed (or at
least that they are not removed after each nucleotide has been
incorporated but are only removed periodically). In other less
preferred embodiments, the primer itself and its extension product
may be removed and replaced with another primer. If required,
several steps of sequential label-free nucleotide additions may be
performed before actual sequencing in the presence of labeled
nucleotides is resumed. A further alternative is to use a different
type of label from that used initially (e.g. by switching from
fluorescein to rhodamine) should quenching effects become
problematic.
In preferred embodiments of the present invention a plurality of
labeled bases are incorporated into an extended primer during
sequencing. This is advantageous in that it can speed up the
sequencing procedure relative to methods in which, once a labeled
base has been incorporated into an extended primer, the label must
be removed before a further labeled base can be incorporated. (The
plurality of labeled bases may be in the form of one or more
contiguous stretches, although this is not essential.)
The present invention therefore also includes within its scope a
method for sequencing nucleic acid molecules, comprising the steps
of: a) using a first colony to provide a plurality of single
stranded nucleic acid molecules that have the same sequences as one
another and that are hybridised to primers in a manner to allow
primer extension in the presence of nucleotides and a nucleic acid
polymerase; b) using a second colony to provide a plurality of
single stranded nucleic acid molecules that have the same sequences
as one another, and that are also hybridised to primers in a manner
to allow primer extension in the presence of nucleotides and a
nucleic acid polymerase; c) providing each colony with a nucleic
acid polymerase and a given labeled nucleotide under conditions
that allow extension of the primers if a complementary base or if a
plurality of such bases is present at the appropriate position in
the single stranded nucleic acid molecules; d) detecting whether or
not said labeled nucleotide has been used for primer extension at
each colony by determining whether or not the label present on said
nucleotide has been incorporated into extended primers; e)
repeating steps c) and d) one or more times so that extended
primers comprising a plurality of labels are provided.
Preferably the sequences of the nucleic acid molecules present at
said first and said locations are different from one another--i.e.
a plurality of colonies comprising different nucleic acid molecules
are sequenced.
In view of the foregoing description it will be appreciated that a
large number of different sequencing methods using colonies of the
present invention can be used. Various detection systems can be
used to detect labels used in sequencing in these methods (although
in certain embodiments detection may be possible simply by eye, so
that no detection system is needed). A preferred detection system
for fluorescent labels is a Charge-Coupled-Device (CCD) camera,
which can optionally be coupled to a magnifying device. Any other
device allowing detection and, preferably, also quantification of
fluorescence on a surface may be used. Devices such as fluorescent
imagers or confocal microscopes may be chosen.
In less preferred embodiments, the labels may be radioactive and a
radioactivity detection device would then be required. Ideally such
devices would be real-time radioactivity imaging systems. Also less
preferred are other devices relying on phosphor screens (Molecular
Dynamics) or autoradiography films for detection.
Depending on the number of colonies to be monitored, a scanning
system may be preferred for data collection. (Although an
alternative is to provide a plurality of detectors to enable all
colonies to be covered.) Such a system allows a detector to move
relative to a plurality of colonies to be analysed. This is useful
when all the colonies providing signals are not within the field of
view of a detector. The detector may be maintained in a fixed
position and colonies to be analysed may be moved into the field of
view of the detector (e.g. by means of a movable platform).
Alternatively the colonies may be maintained in fixed position and
the detection device may be moved to bring them into its field of
view.
The detection system is preferably used in combination with an
analysis system in order to determine the number (and preferably
also the nature) of bases incorporated by primer extension at each
colony after each step. This analysis may be performed immediately
after each step or later on, using recorded data. The sequence of
nucleic acid molecules present within a given colony can then be
deduced from the number and type of nucleotides added after each
step.
Preferably the detection system is part of an apparatus comprising
other components. The present invention includes an apparatus
comprising a plurality of labeled nucleotides, a nucleic acid
polymerase and detection means for detecting labeled nucleotides
when incorporated into a nucleic acid molecule by primer extension,
the detection means being adapted to distinguish between signals
provided by labeled nucleotides incorporated at different
colonies.
The apparatus may also include temperature control, solvent
delivery and washing means. It may be automated.
Methods and apparatuses within the scope of the present invention
can be used in the sequencing of: unidentified nucleic acid
molecules (i.e. de novo sequencing); and nucleic acid molecules
which are to be sequenced to check if one or more differences
relative to a known sequence are present (e.g. identification of
polymorphisms). This is sometimes referred to as
"re-sequencing".
Both de novo sequencing and re-sequencing are discussed in greater
detail later on (see the following sections (v) and (vi)).
For de novo sequencing applications, the order of nucleotides
applied to a given location can be chosen as desired. For example
one may choose the sequential addition of nucleotides dATP, dTTP,
dGTP, dCTP; dATP, dTTP, dGTP, dCTP; and so on. (Generally a single
order of four nucleotides would be repeated, although this is not
essential). For re-sequencing applications, the order of
nucleotides to be added at each step is preferably chosen according
to a known sequence.
Re-sequencing may be of particular interest for the analysis of a
large number of similar template molecules in order to detect and
identify sequence differences (e.g. for the analysis of recombinant
plasmids in candidate clones after site directed mutagenesis or
more importantly, for polymorphism screening in a population).
Differences from a given sequence can be detected by the lack of
incorporation of one or more nucleotides present in the given
sequence at particular stages of primer extension. In contrast to
most commonly used techniques, the present method allows for
detection of any type of mutation such as point mutations,
insertions or deletions. Furthermore, not only known existing
mutations, but also previously unidentified mutations can be
characterised by the provision of sequence information.
In some embodiments of the present invention long nucleic acid
molecules may have to be sequenced by several sequencing reactions,
each one allowing for determination of part of the complete
sequence. These reactions may be carried out at different colonies
(where the different colonies are each provided with the same
nucleic acid molecules to be sequenced but different primers), or
in successive cycles applied at the same colony (where between each
cycles the primers and extension products are washed off and
replaced by different primers).
(iv) DNA Fingerprinting
This embodiment of the present invention aims to solve the problem
of screening a large population for the identification of given
features of given genes, such as the detection of single nucleotide
polymorphisms.
In one preferred embodiment, it consists in generating tagged
genomic DNA (see section G (ii) supra). (Thus each sample
originating from a given individual sample has been labeled with a
unique tag). This tagged DNA can be used for generating primary
colonies on an appropriate surface comprising immobilised primers.
Several successive probe hybridisation assays to the colonies can
then be performed. Between each assay the preceding probe can be
removed, e.g. by thermal denaturation and washing.
Advantages of this embodiment of the present invention over other
approaches for solving this problem are illustrated in the
following example of a potential practical application.
It is intended to detect which part of a gene (of, e.g., 2000 bases
in size), if any, is related to a disease phenotype in a population
of typically 1,000 to 10,000 individuals. For each individual, a
PCR amplification can be performed to specifically amplify the gene
of interest and to link a tag and a colony generating primer (refer
to section G (iv), preparation of "tagged DNA").
In order to obtain a representative array of sample, one might want
to array randomly 500,000 colonies (i.e. 10 times redundancy, so to
have only a small probability of missing the detection of a
sample). With a colony density of 10,000 colonies per mm.sup.2, a
surface of .about.7 mm.times.7 mm can be used. This is a much
smaller surface than any other technology available at present time
(e.g. The HySeq approach uses 220 mm.times.220 mm for the same
number of samples (50,000) without redundancy). The amount of
reactants (a great part of the cost) will be proportional to the
surface occupied by of the array of samples. Thus the present
invention can provide an 800 fold improvement over the presently
available technology.
Using an apparatus to monitor the result of the `in situ`
sequencing or probe hybridisation assays, it should take on the
order of 1 to 10 seconds to image a fluorescent signal from
colonies assayed using fluorescence present on a surface of
.about.1 mm.sup.2. Thus, assuming that the bottleneck of the method
is the time required to image the result of the assay, it takes of
the order of 10 minutes to image the result of an assay on 50,000
samples (500,000 colonies). To provide 200 assays including imaging
(on one or several 7 mm.times.7 mm surfaces), using the present
invention can take less than 36 hours. This represents a 20 times
improvement compared to the best method known at present time
(HySeq claims 30 days to achieve a comparable task).
Improvements (colony densities 10 times higher and imaging time of
1 second) could allow for much higher throughput and finally the
ultimately expected throughput could be about 2000 times faster
than the best, not yet fully demonstrated, technology available at
present time.
Another advantage of using the present invention lies in the fact
that it overcomes the problem arising with individuals who have
heterozygous mutations for a given gene. While this problem may be
addressed by existing sequencing methods to determine allelic
polymorphisms, current high throughput mutation detection methods
based on oligonucleotide probe hybridisation may lead to
difficulties in the interpretation of results due to an unequal
hybridisation of probes in cases of allelic polymorphisms and
therefore errors can occur. In this embodiment of the present
invention, each colony arises from a single copy of an amplified
gene of interest. If an average of 10 colonies are generated for
each individual locus, there will be an average of 5 colonies
corresponding to one version of a gene and 5 colonies corresponding
to the other version of the gene. Thus heterozygotic mutations can
be scored by the number of times a single allele is detected per
individual genome sample.
(v) DNA Resequencing
This embodiment of the present invention provides a solution to the
problem of identifying and characterising novel allelic
polymorphisms within known genes in a large population of
biological samples.
In its preferred embodiment it consists in obtaining tagged DNA
(each sample originating from a given individual has been tagged
with a unique tag--see section G (iv)). This encoded DNA can then
be used for generating primary colonies on an appropriate surface
comprising immobilised primers. Several successive assays of probe
hybridisation to the colonies can then be performed wherein between
each cyclic assay the preceding probe can be removed by thermal
denaturation and washing. Preferably, the DNA sequence 3' to a
specific probe may be determined directly by `in situ sequencing`
(section I (iii)), Methods of sequencing).
The advantages of the present invention over other approaches for
solving this problem are illustrated in the following example of
potential practical application:
It is desired to identify the variability of the sequence of a gene
(of, e.g., 2 000 bases in size), if any, in a population of
typically 4 000 individuals. It is assumed that a reference
sequence of the gene is known. For each individual, PCR
amplification can be performed to specifically amplify the gene of
interest and link a tag and a colony generating primer. In order to
obtain a representative array of sample, one might want to array
randomly 40 000 colonies (i.e. 10 times redundancy, so to have a
small probability of missing the detection of a sample). With a
colony density of 10 000 colonies per mm.sup.2, a surface of
.about.2 mm.times.2 mm can be used.
Using an apparatus with a CCD camera (having a 2000.times.2000
pixel chip) to monitor the result of the assay, it should take of
the order of 10 seconds to image a fluorescent signal from colonies
on a surface of 4 mm.sup.2. If it is assumed possible to read at
least 20 bases during one round of the assay, this requires 61
imaging steps (3n+1 imaging steps are necessary for reading n
numbers of bases). If it is assumed that the bottleneck of the
method is the time to image the result of the assay, it takes of
the order of 15 minutes to image the result of an assay on 4 000
samples (40 000 colonies). To realise 100 assays (on one or several
2.times.2 mm.sup.2 surfaces) in order to cover the entire gene of
interest, the present invention can allow the whole screening
experiment to be performed in approximately one day, with one
apparatus. This can be compared to the most powerful systems
operational at the present time.
In this embodiment of the present invention with conservative
assumptions (colony density, imaging time, size of the CCD chip), a
throughput of 3.2.times.10.sup.6 bases per hour could be reached,
i.e. a 400 fold improvement when compared to the most commonly used
system at present time (current DNA sequencers have a typical
throughput of the order of 8,000 bases read/hour).
(vi) De Novo DNA Sequencing
This embodiment of the present invention aims to solve the problem
of sequencing novel genomes (or parts thereof) with low cost and in
short time, where the sequence of the DNA is not known. Genomic DNA
can be prepared, either directly from the total DNA of an organism
of interest or from a vector into which DNA has been inserted. The
prepared genomic DNA (from whatever source) can be used to generate
DNA colonies. The DNA colonies can then be digested with a rare
cutting restriction enzyme, whose site is included in the linker,
denatured and sequenced.
FIG. 18 depicts an example of de novo DNA sequencing. In this
example, genomic DNA is fragmented into pieces of 100 to 2,000 base
pairs (see preparation of random DNA fragments, section G (i)).
These fragments will be ligated to oligonucleotide linkers (IIa and
IIb, FIG. 18, (a)) which include sequences specific for the grafted
primers on the surface (`P1` and `P2`), a sequence which is
recognised by a rare-cutting restriction nuclease (`RE`) and a
sequence corresponding to a sequencing primer (`SP`), resulting in
templates (III, FIG. 18, (b)). Using this prepared DNA as template
for DNA colony formation, one obtains primary colonies (IV, FIG.
18, (c)). These colonies are then digested with the corresponding
restriction endonuclease and denatured to remove the non-attached
DNA strand (V, FIG. 18, (d)). The sequencing primer (SP) is then
annealed to the attached single-stranded template (FIG. 18, (e)).
Incorporation and detection of labeled nucleotides can then be
carried out as previously described (see section (iii), Methods of
Sequencing).
In this embodiment, the throughput obtainable can be at least 400
times higher than presently available methods.
(vii) mRNA Gene Expression Monitoring
This embodiment of our invention means to solve the problem of
monitoring the expression of a large number of genes
simultaneously.
Its preferred embodiment is depicted in FIG. 19.
Firstly, primary colonies are prepared, as depicted in FIG. 3. In
its preferred form, the DNA used for this preparation is `prepared
genomic DNA` or `tagged genomic DNA`, as described in section G(i)
and G(iii), respectively, and where the DNA is either from the
whole genome of one (or several) organism(s) or from a subset
thereof (e.g., from a library of previously isolated genes). In
FIG. 19, the uppercase letters, "A", "B" and "D" represent colonies
which have arisen from genes which exhibit high, medium and low
expression levels, respectively, and "E" represents colonies
arising from nonexpressed genes (in real cases, all these
situations may not necessarily be present simultaneously).
Secondly, the colonies are treated to turn then into supports (i.e.
secondary primers) for secondary colony growing (step i in FIG. 19,
(a)), as described in section E. At this stage (FIG. 19, (a)), the
treated colonies are represented by underlined characters (A, B, D,
or E).
Thirdly, (step ii in FIG. 19, (b)) this support for secondary
colony growing is used to regenerate colonies from mRNA (or cDNA)
templates extracted from a biological sample, as described in
section C. If the template is mRNA, the priming step of colony
regeneration will be performed with a reverse transcriptase. After
a given number of colony amplification cycles, preferably 1 to 50,
the situation will be as depicted in (FIG. 19, (c)): the colonies
corresponding to highly expressed genes (represented by the letter
"A") are totally regenerated, as their regeneration has been
initiated by many copies of the mRNA; the colonies corresponding to
genes of medium expression levels (represented by the letters "b"
and "B"), have been only partially regenerated; only a few of the
colonies corresponding to rare genes (represented by the letter
"d"), have been partially regenerated; the colonies corresponding
to non-expressed sequences (represented by the letter "E"), have
not been regenerated at all.
Lastly, (step iii in FIG. 19, (c)), additional cycles of colony
growing are performed (preferably 2 to 50), and the colonies which
have not been totally regenerated during the previous steps finally
become totally regenerated, "b" becomes "B", "d" becomes "D" (FIG.
19, (d)): the colonies corresponding to genes with high and medium
expression levels are all regenerated "A" and "B" or "B"; the
colonies corresponding to genes with low levels of expression are
not all regenerated "D" and "D"; the colonies corresponding to
non-expressed sequences are not regenerated at all "E".
The relative levels of expression of the genes can be obtained by
the following preferred methods: Firstly levels of expression can
be monitored by following the rate of regeneration of the colonies
(i.e., by measuring the amount of DNA inside a colony after
different number of colony growing cycles during step (iii)) as the
rate at which a colony is regenerated will be linked the number of
mRNA (or cDNA) molecules which initiated the regeneration of that
colony (at first approximation, the number of DNA molecules after n
cycles, noted M(n), in a colony undergoing regeneration should be
given by M(n)=M.sub.0r.sup.(n-1), where M.sub.0 is the number of
molecules which initiated the regeneration of the colony, r is the
growing rate and n is the number of cycles); Secondly, levels of
expression can be monitored by counting, for each gene, the number
of colonies which have been regenerated and comparing this number
to the total number of colonies corresponding to that gene. These
measurements will generally give access to the relative expression
levels of the genes represented by the colonies. The identification
of the colonies is preferably performed by fingerprinting, in a
manner essentially similar to embodiment, section I(iv). Note that
encoding the DNA samples is not required, but can be considered as
an alternative to the direct identification of the DNA in the
colonies. This can be of practical interest because with coding,
the same codes (thus the same oligonucleotides involved in assaying
the code) can be used for any set of genes, whereas without code, a
different set of specific oligonucleotides has to be used for each
set of genes.
This embodiment of our invention has many advantages if compared to
current state of the art including: a very high throughput; no
requirement for prior amplification of the mRNA (even though prior
amplification is compatible with or invention); small amounts of
samples and reactants are required due to the high density of
samples with our invention; the presence of highly expressed genes
has no incidence on the ability to monitor genes with low levels of
expression; the ability to simultaneously monitor low and high
levels of expression within the set of genes of interest.
When the initial DNA in the generation of the primary DNA colony is
made from the DNA of a whole genome, this embodiment also provides
the following features: there is no interference between genes
expressed at high level and at low level even though one has not
performed specific amplification of the genes of interest. This is
a unique feature of the use of this invention: specific
amplification is not possible because the initial assumption of
this embodiment is to monitor the expression genes which may have
not yet been isolated, thus which are unknown, and thus for which
no specific (unique) sequences are known and which specific
sequences would have been necessary for specific gene
amplification. The ability of our invention to perform this type of
mRNA expression monitoring is due to the fact that when the primary
colonies are prepared, statistically, each piece of the initial
genome will be represented by the same number of colonies. Thus,
frequent and rare DNA will initiate the same number of colonies
(e.g., one colony per added genome molecule). Quantitative
information might be obtained both from frequent and rare mRNAs by
monitoring the growing rate of the colonies.
(viii) Isolation and Characterisation of Novel Expressed Genes
This embodiment of our invention means to solve the problem of
isolating the genes which are specifically induced under given
conditions, e.g., in specific tissues, different strains of a given
species or under specific activation. A practical example is the
identification of genes which are up or down regulated after drug
administration.
The preferred embodiment for isolating genes from a specific or
activated biological sample (hereafter called target sample) which
are up-regulated compared with a reference biological sample
(hereafter called reference sample) is depicted in FIG. 20.
Firstly, primary colonies are prepared (FIG. 20, (a)). In its
preferred form, the DNA used for this preparation is prepared
genomic DNA or tagged genomic DNA, as described in sections G(i)
and G(ii), respectively, where the DNA is either from the whole
genome of one (or several) organism(s) or from a subset thereof
(e.g., from a library of previously isolated genes), and where both
the primers used for colony generation (hereafter called P1 and P2)
contain a endonuclease restriction site. In FIG. 20 (a), "A"
represents colonies which have arisen from genes expressed in both
the reference sample and the target sample, "B" represents colonies
which have arisen from genes expressed only in the reference
sample, "C" represents colonies which have arisen from genes
expressed only in the target sample, and "D" represents colonies
arising from non-expressed genes (in real cases, all these
situations may not necessarily be present simultaneously).
Secondly, primary colonies are then treated to generate secondary
primers as the support for secondary colony growing (step i in FIG.
20(a)). At this stage (b), the colonies are represented as
underlined characters (A, B, C, D).
Thirdly, (step ii in FIG. 20, (b)) the secondary primers are used
to regenerate colonies using mRNA or cDNA (represented by "mA+mB")
extracted from the biological reference sample as a template, as
described in G(v). If the template is mRNA, the first elongation
step of colony regeneration will be performed with a reverse
transcriptase. After enough colony growing cycles, preferably 5 to
100, only the colonies corresponding to genes expressed in the
reference sample ("A" and "B") will be regenerated, as depicted in
(FIG. 20, (c)).
In step (iii), the colonies are digested with a restriction enzyme
(represented by RE) which recognises a site in the flanking primer
sequences, P1 and P2, which are grafted on the support and which
were the basis of primary colony generation. Importantly, only the
colonies which have been regenerated during step (ii) will be
digested. This is because the support for secondary colony growth
is made of single stranded DNA molecules, which can not be digested
by the restriction enzyme. Only the regenerated colonies are
present in a double stranded form, and are digested. After
digestion, the situation is the one depicted in FIG. 20 (d). The
colonies corresponding to the genes expressed in the reference
sample have totally disappeared, i.e., they are not even present as
a support for secondary colony growth, and the colonies
corresponding to genes expressed only in the target sample "C" and
the colonies corresponding to non-expressed genes "D" are still
present as a support for secondary colony generation.
In step (iv), mRNA (or cDNA) (represented by "mA+mC") extracted
from the target sample is used to generate secondary colonies.
Because colonies corresponding to mA and mB no longer exist, only
the colonies corresponding to mC can be regenerated (i.e., only the
mRNA specifically expressed in the target sample). After sufficient
number of colony growing cycles (preferably 5 to 100), the
situation is such that only the colonies corresponding to genes
expressed specifically in the target sample are regenerated ("C",
FIG. 20, (e)).
In step (v), the regenerated colonies "C" are used to generate
copies of the DNA that they contain by performing several
(preferably 1 to 20) colony growing cycles in the presence of the
primers P1 and P2, as described in section D of the present
invention. A PCR amplification is then performed using P1 and P2 in
solution (described in section D) and the amplified DNA
characterised by classical methods.
The preferred embodiment for isolating genes from a specific or
activated biological sample which are less expressed than in a
reference biological sample is depicted in FIG. 21. The different
steps involved in this procedure are very similar to those involved
in the isolation of gene which are more regulated than in the
reference sample, and the notation are the same as in FIG. 20. The
only difference is to inverse the order used to regenerate the
colonies: in step (ii), the mRNA used is the one extracted from the
target biological sample ("mA+mC") instead of the mRNA extracted
form the reference biological sample ("mA+mB"), and in step (iv),
the mRNA used is the one extracted from the reference biological
sample ("mA+mB") instead of the one extracted from the target
sample ("mA+mC"). As a result, only the DNA from colonies
corresponding to genes which are expressed in the reference sample
but not in the target sample is recovered and amplified ("B", FIG.
21, (f)).
SEQUENCE LISTINGS
1
1630 base pairsnucleic acidsinglelinearDNA 1TTTTTTCACC AACCCAAACC
AACCCAAACC 3030 base pairsnucleic acidsinglelinearDNA 2TTTTTTAGAA
GGAGAAGGAA AGGGAAAGGG 3024 base pairsnucleic acidsinglelinearDNA
3AGAAGGAGAA GGAAAGGGAA AGGG 2424 base pairsnucleic
acidsinglelinearDNA 4CCCTTTCCCT TTCCTTCTCC TTCT 2420 base
pairsnucleic acidsinglelinearDNA 5GCGCGTAATA CGACTCACTA 2020 base
pairsnucleic acidsinglelinearDNA 6CGCAATTAAC CCTCACTAAA 2026 base
pairsnucleic acidsinglelinearDNA 7TTTTTTCTCA CTATAGGGCG AATTGG 2630
base pairsnucleic acidsinglelinearDNA 8TTTTTTCTCA CTAAAGGGAA
CAAAAGCTGG 3034 base pairsnucleic acidsinglelinearDNA 9TTTTTTTTTT
CACCAACCCA AACCAACCCA AACC 3434 base pairsnucleic
acidsinglelinearDNA 10TTTTTTTTTT AGAAGGAGAA GGAAAGGGAA AGGG 3444
base pairsnucleic acidsinglelinearDNA 11CACCAACCCA AACCAACCCA
AACCACGACT CACTATAGGG CGAA 4444 base pairsnucleic
acidsinglelinearDNA 12AGAAGGAGAA GGAAAGGGAA AGGGTAAAGG GAACAAAAGC
TGGA 4420 base pairsnucleic acidsinglelinearDNA 13GGTGCTGGTC
CTCAGTCTGT 2020 base pairsnucleic acidsinglelinearDNA 14CCCGCTTACC
AGTTTCCATT 2020 base pairsnucleic acidsinglelinearDNA 15CTGGCCTTAT
CCCTAACAGC 2020 base pairsnucleic acidsinglelinearDNA 16CGATCTTGGC
TCATCACAAT 20
* * * * *